CN105051537B - Cell-mediated immune response test - Google Patents

Cell-mediated immune response test Download PDF

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Publication number
CN105051537B
CN105051537B CN201380065249.4A CN201380065249A CN105051537B CN 105051537 B CN105051537 B CN 105051537B CN 201380065249 A CN201380065249 A CN 201380065249A CN 105051537 B CN105051537 B CN 105051537B
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antigen
cell
sample
disease
composition
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CN105051537A (en
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J·伯勒
A·奈茨
C·穆尼安
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Cellestis Ltd
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Cellestis Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma

Abstract

Present disclosure relates generally to based on immunologic diagnostic test field, and described based on immunologic diagnostic test includes the cell-mediated immune response power test of detection.Present invention teaches the exposure based on cell-mediated immune response diagnosis object to antigen, there is the sensitivity of enhancing, and by realizing in sample with antigen incubation period addition non-reducing sugar.

Description

Cell-mediated immune response test
Technical field
The present invention relates generally to immunologic test field is based on, and describe for measuring cell-mediated immune response The method of power.Present invention teaches for measuring the active method of cell-mediated immune response, composition and kit, have There are the sensitivity and improved storage stability of enhancing.
Background of invention
Had based on immunologic diagnostic test in medical field and is widely applied.Specifically, it is auxiliary detection and prison Survey the important tool of a variety of disease symptoms.The effect portion of these test types is component in immune system, and (such as T lymph is thin Born of the same parents) specificity.In spite of such specificity, but in active or latent infection morbid state object or performance Out detection infection in immune deficiency or any type of immunosuppressive object, detect whether that there are rudimentary infection (to continue low water Flat infection) for, it is always not sensitive enough based on immunologic diagnosis.The required performance of cell-mediated immune response test Feature, for detection antigen-specific T cell response, including suitable sensitivity, specificity, reliability and again Existing property, additionally should simply and fastly be operated.
It is a kind of established to be related to based on immunologic diagnostic test form using antigenic stimulus T cell or immune system Other cells, the subsequent immune effector molecule (such as IFN-γ or other cell factors) for detecting response antigenic stimulus and generating.Make Immune effector molecule is detected with well known technology, such as Dot Enzyme Immunoassay, the analysis of polynary pearl, ELISA, ELISpot and fluidic cell Art.Immune effector molecule can be also measured based on rna level whether there is or its horizontal raising.This kind of test can for example be used to examine Survey disease-specific immune response, especially pathogen specific immune response.Each test is in trade mark QuantiFERON (registration Trade mark, the Leix Sai Er Di Si Co., Ltd (Cellestis Limited)) under it is commercially available and can for example be used to diagnose pathogen Infection or monitoring are immunized for the cell-mediated of disease.
The other application of each cell-mediated immune response test includes that analysis or monitoring are directed to vaccine or immunization therapy (such as Cancer immunotherapy) cellullar immunologic response.
It is highly desirable to each test of the sensitivity with enhancing.Previously, by the way that sample (such as whole blood sample) and antigen exist Be incubated in the presence of monosaccharide (such as dextrose) improve for measuring cell-mediated immune response method (referring to Such as 2004/042396 A1 of WO).It has been found that monosaccharide (such as dextrose and glucose) increases the IFN- generated by immunocyte γ and the sensitivity for thus improving test.For allow cell using the energy source and therefore benefit from antigen incubation period For addition sugar, the use of monosaccharide (such as dextrose and glucose) is considered necessary.In except antigen extroversion sample directly When adding monosaccharide, the significant raising of INF- γ level is observed.However, preferably providing instant examination to simplify the implementation of method Agent composition simultaneously avoids manual operation step.Accordingly, it is desirable to provide the single composition comprising antigen and monosaccharide.The composition It can be provided in sample collection tube, wherein sample directly contacts antigen and monosaccharide with correct concentration, to avoid operating error.So And find herein, when each sample collecting pipe comprising antigen and monosaccharide stores at room temperature or at raised temperature, examination Activity is tested to reduce at any time.When this is found in using certain antigens.Therefore, the storage phase of these reagent constituents is limited, and After one section of storage time, which only provides muting sensitivity or even test activity completely loses.Therefore, each kit/examination Testing material (wherein easily providing antigen and monosaccharide in a composition) is not stable storing, and therefore has test spirit The risk that sensitivity is reduced at any time or even lost.When not including monosaccharide and antigen in sample collection tube, but monosaccharide individually adds When adding to sample to be incubated for antigen, above-mentioned phenomenon is not observed.
The purpose of the present invention is overcome at least one defect of art methods.Specifically, the purpose of the present invention is Sensitive and reliable method is provided to measure the kit and reagent constituents of cell-mediated immune response and stable storing.
Summary of the invention
Inventors have found that unexpectedly increasing response level simultaneously in sample and antigen incubation period addition non-reducing sugar Therefore the sensitivity of this method is improved, is similar to and is added seen in monosaccharide (such as dextrose and glucose) now in incubation period As.This is beyond expection, because it was previously believed that monosaccharide only can be used and therefore realized using the monosaccharide for representing reduced sugar The raising of sensitivity.In addition, being also unexpectedly found that, this method can also be maintained even if during the storage of extended test material Its increased sensitivity, even if being also such when providing non-reducing sugar and antigen in the form of single composition.Therefore, it invents People it was unexpectedly found that, using non-reducing sugar replace monosaccharide significantly increase test component storage stability, he improves simultaneously Response is horizontal and therefore can improve assay sensitivity.It is without being bound by theory, it is believed that non-reducing sugar is mediated in positive cell The amount of the immune effector molecule of release is increased in the case where immune response, so as to improve assay sensitivity.Obviously, immunological effect Molecule, which generates, to be enhanced.Therefore, it is also monitored earlier than other possible methods using non-reducing sugar as described herein Immunocyte stimulation.The ability for improving the sensitivity of cell-mediated immune response test can also promote using compared with muting sensitivity Effector molecule monitoring method and/or the lesser sample size of use.In addition, these beneficial effects can also be stored up in extended test material It is obtained after depositing, so as to improve the reliability and reproducibility of test.
According in a first aspect, provide for measuring the active method of cell-mediated immune response, the method includes
(a) it is incubated by making the sample comprising immunocyte contact at least one antigen and at least one non-reducing sugar to provide Composition is educated, the immunocyte can generate immune effector molecule after being stimulated by antigen, and
(b) detect whether that there are immune effector molecule or its levels.
Usable the method is for example with immune response activity cell-mediated in measurement object (such as patient).In the presence of (or There is no) immune effector molecule that detects or its level indicate that object generates cell-mediated immune answer for the antigen of test The level or ability answered.
According to second aspect, the composition of the immune response mediated for inducing cell is provided, the composition includes
A) at least one antigen；
B) at least one non-reducing sugar；And
C) optional at least one anticoagulant.
Composition can respectively be advantageously used in method described in the first aspect of the present invention by making comprising immune thin The sample of born of the same parents contacts the composition to prepare incubation composition.
According to the third aspect, a kind of collection containers are provided, it includes the combinations described in the second aspect of the present invention Object.The collection containers are convenient in method described in the first aspect of the present invention.Preferably, which holds Device is the blood collection tube of vacuum.
According to fourth aspect, provide for the active kit of immune response cell-mediated in measurement object, packet Containing at least one antigen, at least one non-reducing sugar, at least one collection containers (it is preferably blood collection tube) and extremely A kind of few detection means at least one immune effector molecule.Kit can be respectively for progress the first aspect of the present invention The method.
According to the 5th aspect, the present invention relates to non-reducing sugars for measuring the active immunity test of cell-mediated response In application, wherein the addition of non-reducing sugar increases at least one of immunocyte immune effector molecule (preferably IFN-γ) Release, the immunocyte generates response to the antigen tested in the test.
According to following the description and appended claims, other purposes, feature, advantage and aspect of the invention are to this field skill It is obvious for art personnel.However, it should be understood that although following the description, appended claims and specific embodiment indicate The preferred embodiment of the application, also only provides by way of illustration.By reading hereafter, the invention disclosed is fallen in It would have been obvious for a person skilled in the art for different change and modification in spirit and scope.
Brief Description Of Drawings
Influence of the addition trehalose for IFN-γ response in Fig. 1: QFN-CMV test.
(**p<0.01；***p<0.001)
Influence of the addition trehalose for IFN-γ response in Fig. 2: QFN-TB test.
Fig. 3: four kinds of different non-reducing sugars increase quantitative QFN CMV result.
Detailed description of the invention
In the whole text in specification, other explanation unless the context requires, otherwise, term " includes " or its variant such as "comprising" or " containing " be interpreted as the group that instruction includes the element or integer or method and step or element or integer or method and step and It is not excluded for the group of any other element or integer or method and step or element or integer or method and step.
Singular used in this specification "one", "an" and " should (described) " include plural referents, unless up and down Text is expressly stated otherwise.Thus, for example, referring to that " T cell " includes single T cell and two or more T cells；It refers to " anti- It is former " it include single antigen and two or more antigens.Equally, " agent " is referred to, " reagent ", " molecule " and " compound " includes single The combination of a entity and two or more this kind of entities.Refer to that " disclosure " includes the single or multiple sides instructed by present disclosure Face；Deng.The various aspects instructed herein include by term " invention ".All aspects of the invention are all in present claims allowed band It is interior.
According in a first aspect, provide for measuring the active method of cell-mediated immune response, the method includes
(a) it is incubated by making the sample comprising immunocyte contact at least one antigen and at least one non-reducing sugar to provide Composition is educated, the immunocyte can generate immune effector molecule after being stimulated by antigen, and
(b) detect whether that there are immune effector molecule or its levels.
This document describes the Advantageous embodiments of the method and applications.According to first aspect embodiment, mention It has supplied for the active method of immune response cell-mediated in measurement object, the method includes
(a) it is incubated by making the sample comprising immunocyte contact at least one antigen and at least one non-reducing sugar to provide Epidemic disease composition is educated, the immunocyte can generate immune effector molecule after the antigen by being obtained from object is stimulated, and
(b) detect whether that there are immune effector molecule or its horizontal raisings.
Indicate that the cell-mediated of the object immune answers in the presence of (or being not present) immune effector molecule or its horizontal raising Answer horizontal or ability.Specifically, whether the method allows to measure contacted the anti-of antigen or test before the object Antigen representated by original.It immune is answered thus, it is possible to measure the object and whether can cause for the cell-mediated of the antigen It answers.In some embodiments, the quantitative level of cell-mediated immune response can also be measured.In some embodiments, originally The magnitude of the cell-mediated immune response detected in literary disclosed test may be with morbid state, progress and/or seriousness phase It closes.Therefore, the present invention provides the methods of immune response cell-mediated in measure object.The method be able to carry out and/or It supports (but being not limited to) medical diagnosis on disease, especially infectious diseases, pathological condition, allow to measure immunocompetence level and allows to comment Immune cell responses of the valence to endogenous or exogenous agents and albumen toxic agent.The test, which can also be screened or be monitored, is previously exposed to spy Determine the object of antigen (such as antigen associated with disease, infection or pollutant).Other for being described below the method are heavy Want application and use.
It will be explained in detail the single method step and preferred embodiment of the first aspect of the present invention the method now.
Step (a)
In step (a), the sample comprising immunocyte is made to contact at least one antigen and at least one non-reducing sugar, institute Immune effector molecule can be generated after being stimulated by antigen by stating immunocyte.The immunocyte and/or entire sample can obtain The object to be determined from such as its cell-mediated immune response.
Refer to " object ", including such as mankind or non-human species, including primate, livestock animals (such as sheep, ox, pig, Horse, donkey, goat), laboratory test animal (such as mouse, rat, rabbit, cavy, pig, hamster), companion animals (such as dog, cat), bird Class species (such as family's bird, flying bird), reptile and amphibian.Method disclosed herein is in research, physianthropy and poultry It herds, there is practicability in animal doctor and wild application.Preferably, which is people and cell-mediated immune response as described herein Method is for screening to pathogenic microorganisms, virus and the response of helminth power, disease symptom, disease development potentiality, or monitoring The response of tumor challenge or immunization therapy, monitoring immune are answered for the cell-mediated of disease from Immunity, monitored object It answers and for determining whether that there are any immune deficiency or immunosupress.The latter can be by for example including the certain of various chemotherapy agents Drug causes.Alternatively, exposure of the method for the invention measurement to environment albumen toxic agent and pollutant can be used.
The sample includes the immunocyte that immune effector molecule can be generated after using appropriate antigenic stimulus.It is " immune thin Born of the same parents " include but is not limited to lymphocyte (including natural kill (NK) cell, T cell, B cell, macrophage and monocyte), Dendritic cells can generate appointing for one or more immune effector molecules in the response to direct or indirect antigenic stimulus What his immunocyte.Preferably, which includes lymphocyte, more preferably includes T lymphocyte.Term " T cell " and " T lymphocyte " is used interchangeably herein.T cell can cause strong immune response, on condition that the antigen that its identification provides. If antigen representated by the antigen of engaged test or the antigen of test, occurs having the specific note of the antigen before T cell The T cell recalled stimulates rapidly again.These T cells with antigenic specificity are by secretory immune effector molecule (for example, especially interfering Plain γ) carry out response.Then, interference can be measured for the form of the specific marker object of the immune response of the antigen of test Plain γ generates response and the immune effector molecule that discharges for the interferon gamma of release.Therefore, according to one embodiment, should Sample includes T lymphocyte, preferably CD4+T helper cell and/or CD8+Cytotoxic T cell.Preferably, which also includes Corresponding stimulator cell, especially can be by the antigen presenting cell of the antigen presentation of test to T cell.However, can also be independent Suitable antigen presenting cell is added into incubation composition in ground.The antigen presenting cell (APC) added respectively include naturally with And artificial antigen presenting cell or particle.For example, stimulator cell is (such as the matched antigen presentation of self or HLA via radiation Cell) it is optionally separately added to be incubated in composition, the incubation composition then presents antigens to T cell.The reality The mode of applying is for example feasible, on condition that the sample does not include each stimulator cell necessary to inducing T cell response.It is artificial anti- It includes but is not limited to particle relevant to recombination MHC molecule or peptide and recombination costimulatory molecules or lipid capsule that original, which presents embodiment, Bubble.
Preferably, the sample is available from object.According to one embodiment, the sample be comprising immunocyte body fluid or Person is containing the immunocyte from the part of each body fluid.According to preferred embodiment, which is whole blood." whole blood " refers to Substantially undiluted or classification the blood from object.According to one embodiment, which is peripheral blood.However, right For measuring cell-mediated immune response activity, whole blood is preferred and most convenient sample, it is possible to use other contain The sample of immunocyte.Example includes but is not limited to lymph, cerebrospinal fluid, tissue fluid (such as marrow or thymus gland liquid) and breathing liquid (including nose liquid and lung liquid and BAL fluid).The part of above-mentioned sample or derivative (such as consume non-measured cell The sample of cell necessary to the immune response of mediation) it also is used as sample and can be obtained by sample treatment.For example, can pass through Methods known in the art handle whole blood to go unless component necessary to CMI response (such as erythrocyte and/or blood platelet) or Whole blood can be handled with enrichment of leukocytes.Also buffy coat cell or peripheral blood mononuclear cells can be obtained by methods known in the art (PBMC) and sample can be used as.According to one embodiment, the immunocyte of culture is used as sample.In addition, low temperature is protected The cell (the PBMC cell of such as cryo-conservation) deposited also is used as the immunocyte source of object and accordingly acts as sample.For example, The PBMC cell melted contact culture medium can be made to provide the sample containing immunocyte, then make its contact preferably with single group The antigen and non-reducing sugar of solvate form addition are simultaneously incubated for it.According to one embodiment, which exempts from comprising mediated cell All immunocytes necessary to epidemic disease response.However, as described above, individually adding stimulator cell (especially antigen presentation Cell) it is intended to be included within the scope of the present invention.According to one embodiment, the sample include at least T cell (T lymphocyte) and NK cell (NK lymphocyte).According to one embodiment, before contacting the sample with antigen and/or non-reducing sugar, to sample Dilution is no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 3%.
Sample to be analyzed is set to contact at least one antigen and at least one non-reducing sugar to provide incubation composition.
Terms used herein " antigen " refers specifically to can to stimulate or immune response is stimulated (and can especially to stimulate or again again Stimulate cellullar immunologic response) any molecule or reagent.Therefore, term " antigen " can be used in a broad sense.The term is specific Refer to any point for being combined by major histocompatibility complex (MHC) and T cell receptor being presented to or be selectively bound by the antibody Son or reagent.The term also refer to can by non-classical MHC albumen (such as CD1d or other CD1 family members) combine any molecule or Reagent.According to one embodiment, which is immunogene.According to one embodiment, which is not immunogene.Antigen packet Include but be not limited to peptide, albumen, haptens, allergen or toxin or any naturally-produced or molecule of synthesis or part thereof.According to One embodiment, the antigen are inactive pathogen or part thereof or lysate.According to one embodiment, which is selected from The segment of peptide, albumen (including glycoprotein), carbohydrate, phosphatide, phosphoprotein, phospholipoprotein and aforementioned substances.Unless up and down Civilization truly has different explanations, and terms used herein " peptide " further includes polypeptide and albumen.Term " albumen " further includes the shape of modification Formula, such as glycoprotein and phosphoprotein.According to one embodiment, which includes the peptide of one or more overall lengths or partial-length. According to one embodiment, which is provided by peptide.According to one embodiment, the length of one or more peptides as antigen Selected from 5-100 amino acid, preferably 7-50 amino acid.According to one embodiment, the antigen by from it is one or more not The peptide group of same overall length or partial-length provides.Peptide group includes at least two peptides and in one embodiment includes a series of heavy Folded or non-overlap peptide.Each peptide group can cover the whole length or a part of naturally-produced proteantigen.However, the peptide need not Need to be overlapped or can be overlapped by single amino acids or multiple amino acid.According to one embodiment, one kind has been used Peptide group comprising the 80-100% of naturally-produced peptide or protein antigen.
According to one embodiment, the antigen is by least one by CD8+The peptide of cytotoxic T cell identification provides.For The embodiment, the antigen is preferably by less than 15 amino acid of at least one length (preferably 13 amino acid or less, 12 ammonia Base acid or less, 11 amino acid or less or 10 amino acid or less) peptide provide.By CD8+Cytotoxic T cell The suitable magnitude range of the various peptides of identification includes 7-14 amino acid residue, 7-13 amino acid residue, 8-12 amino Sour residue, 8-11 amino acid residue and 8-10 amino acid residue.It also can be used comprising by CD8+Cytotoxic T cell identification Peptide or the peptide group that is made from it.The peptide may include all or part of proteantigen (such as naturally-produced proteantigen). It was found that being shown in the presence of monosaccharide (such as glucose or dextrose) during storage comprising each small peptide as the test of antigen Test active significant decrease.Using the phenomenon is not observed when non-reducing sugar, as present invention teach that as.Herein, Due to incorporating non-reducing sugar, assay sensitivity maintains to increase, even if being also such after extended test component storage time. In use comprising by CD8+The peptide antigen and non-reducing sugar of cytotoxic T cell identification are (such as respectively as kit compound Component) composition when, this is important advantage.
According to one embodiment, which is provided by least two groups peptide, and first group is about 7- comprising at least one length The peptide of 14 amino acid residues and second group are 15 amino acid residues or longer peptide comprising at least one length comprising complete Portion or a part of proteantigen.Each group includes at least one to a series of peptide of overlapping or non-overlap.It will derive from or correspond to Represent the proteantigen of disease or illness to be tested 7-14 amino acid peptide and more than or equal to 15 amino acid peptides be included in Immunocyte in sample is incubated for the relatively sensitive test of generation altogether, immune so as to detect earlier than other possible methods Cell, particularly lymphocyte stimulation.The ability for improving cell-mediated immune response assay sensitivity advantageously reduces detection Limit and/or allow to detect effector cell using less sensitive method.Therefore, by the various peptides of at least two groups and non-reducing sugar Combination is beneficial.It is not bound by the limitation of opinion or binding mode, it is believed that two groups of peptides (7-14 mer peptides and are more than or equal to 15 aggressiveness Peptide) promote CD4+And CD8+The detection of T cell.CD4+T cell identification is greater than 15 mer peptides and CD8+T cell identifies 7-14 aggressiveness Peptide.These peptides are referred to herein as " CD4+Peptide " (being more than or equal to 15 mer peptides) or " CD8+Peptide " (7-14 mer peptides).Each group includes At least one peptide and in one embodiment include a series of overlappings peptide.Therefore, can be containing a series of length for first group The peptide of the overlapping of 7-14 amino acid residue.These peptides are by CD8+T cell identifies (CD8+Peptide).Second group can contain a series of length Degree is greater than the peptide of the overlapping of 15 amino acid residues.These peptides are by cytotoxicity CD4+T cell identifies (CD4+Peptide).Two groups of peptides are all The whole length or a part of proteantigen are covered, such as represents the naturally-produced proteantigen of disease or illness to be tested. It is that the peptide needs not be overlapping or can be overlapped by single amino acids or multiple amino acid.The peptide includes peptide group (pods of Peptides) comprising and therefore cover the 80-100% of proteantigen." 80-100% " refer to 80,81,82,83,84,85, 86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%.Refer to according to one embodiment including all Or the length of a part of proteantigen refers to that length is about 7 ammonia when being a series of peptide of overlappings of about 7-14 amino acid residue Base acid residue to most 14 amino acid residues peptide or part thereof, the peptide from the N-terminal of proteantigen to its C-terminal in total It crosses over from each amino acid residue of proteantigen to most 6 amino acid residues.Therefore, if the length of given peptide is x ammonia Base acid residue, wherein x is about 7-14, then the length being overlapped between two kinds of continuous peptides is x-1 to x-6.In one embodiment, Each continuous peptide is laminated in x-1.The peptide that length is more than or equal to a series of overlappings of 15 amino acid residues also crosses over whole or portion Divide proteantigen, wherein the length of each peptide is the length of at least 15 amino acid residues or most whole protein antigens.At one In embodiment, the peptide that length is more than or equal to 15 amino acid residues is about 15-50 amino acid, such as 15,16,17,18,19, 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、 45,46,47,48,49 or 50 amino acid residues.
The present invention includes following situations, i.e., series or peptide group in each peptide be equal length (i.e. x).However, the peptide system Column or peptide group may include x1、x2、xj…xiThe mixture of peptide, wherein each x according to one embodimentiThe length of peptide is about 7-14 Amino acid residue is more than or equal to 15 amino acid residues.The CD4+And/or CD8+Peptide can be divided into individual peptide pond.It can be independent Ground is added in sample or may include in a composition, preferably together with non-reducing sugar.The composition can be then set to contact sample Product are to prepare incubation composition.
In some embodiments, one or more antigens have been used, have been in the antigen for being handed to immune system in analogue body One or more effects.According to one embodiment, which is selected from autoantigen, from the antigen from causal organism Or immune response or tumor associated antigen with the stimulation of its antigen with cross reaction, metal or inorganic molecule.According to one Embodiment, the antigenic source in the antigen from pathogen relevant to disease symptom and therefore with it with cross reaction, Perhaps the antigen is tumor associated antigen relevant to cancer or the antigen is or from toxic agent.According to an embodiment party Formula, using sample and specificity for the disease of the immune response mediated for test cell or the antigen contact of illness, such as Antigen that is related to disease to be assessed or illness or representing disease or illness to be assessed.According to one embodiment, which is Disease-specific antigens, specifically pathogen specific antigen.In some embodiments, which is bacterium, disease Poison, helminth or fungi.In an illustrated embodiment, the antigen is from mycobacteria (mycobacterium) Antigen specifically comes from the antigen of mycobacterium tuberculosis (Mycobacterium tuberculosis).Therefore, some In embodiment, which is tuberculosis (TB) specific antigen.For example, the antigen can be from mycobacterium tuberculosis or bird point The protein derivatives of the purifying of branch bacillus (M.avium).In some embodiments, the antigenic stimulus Mycobacterium proteins, such as ESAT-6, CFP-10 and TB7, respectively TB7.7.In another illustrated embodiment, which comes from or specific needle To virus, such as cytomegalovirus (CMV).
Then also describe other examples of antigen, particularly disease-specific antigens.
In addition, sample contacts non-reducing sugar in step a)." non-reducing sugar " refer specifically to not be directed to reduced sugar detection Reagent (such as Fehling's solution (Fehling's solution), benedict's reagent (Benedict's reagent) or support human relations Reagent (Tollens'reagent)) sugar that reacts.Non-reducing sugar does not include free reduction end and does not therefore include free Aldehyde radical or free ketone group.The non-reducing sugar can have any length and can be linear chain or branched chain.In some embodiments, The non-reducing sugar contains at least two monosaccharide unit.According to one embodiment, in any and whole monosaccharide list of non-reducing sugar In member, the carbon atom adjacent with the oxygen atom in ring structure does not include hydroxyl and does not therefore include different head hydroxyl.According to a reality Mode is applied, the ring structure of the monosaccharide unit of the non-reduced oligosaccharides does not include hemiacetal or hemiketal group.According to an embodiment party Formula, the non-reducing sugar are oligosaccharides, it includes 10 monosaccharide units or less, more select 8 monosaccharide units or less, 6 monosaccharide Unit or less, 5 monosaccharide units or less, 4 monosaccharide units or less, 3 monosaccharide units or less or 2 monosaccharide lists Member.Preferably, which is disaccharides.According to one embodiment, glycosidic bond is formed between monosaccharide unit, connection one The reduction end of a monosaccharide unit and the reduction end of another monosaccharide unit.The preferred example of non-reducing sugar is sucrose and sea Algae sugar.In addition, as shown in the Examples, mannitol and gossypose also are used as non-reducing sugar.Therefore, according to one embodiment, The non-reducing sugar is selected from trehalose, mannitol, sucrose and gossypose.As shown in the Examples, these exemplary non-reducing sugars improve The magnitude of response.Trehalose is particularly preferred, because experiment display trehalose improves the magnitude of response and therefore improves examination Sensitivity is tested, the composition furthermore comprising trehalose and antigen shows outstanding storage stability.As shown in the Examples, it is testing Non-reducing sugar in, using when trehalose response raising act on it is most strong.However, the non-reducing sugar can also be monosaccharide, wherein example Such as, reduction end is coupled with another chemical entities and thereby is hindered by it.Therefore, which can be derivatization.Sugar The example of derivative is amino sugar, wherein one or more hydroxyls are replaced by amino or acetylamino.In preferred embodiment In, which is unsubstituted and especially underivatized.According to one embodiment, which is not more Sugar.In some embodiments, which is not associated with to albumen, peptide or lipid or other macromoleculars.Implemented according to one Mode, the non-reducing sugar are not included in cell culture medium or other culture mediums.According to one embodiment, the non-reducing sugar is not Comprising in a liquid.The non-reducing sugar is can be metabolized by the immunocyte for including in sample.According to one embodiment, when this When non-reducing sugar is present in the incubation composition comprising sample and antigen with debita spissitudo, it is thin that the T stimulated again can be increased The interferon gamma that born of the same parents are discharged.
By contacting the sample with antigen, non-reducing sugar and other optional additives, incubation composition is provided.It is preferred that More than room temperature and therefore ground, the mixtures incubated are incubated at elevated temperatures.Preferably, which is above 30 DEG C, preferably higher than 35 DEG C.Suitable incubation temperature range includes 30 DEG C to 40 DEG C, preferably 35 DEG C to 40 DEG C.Advantageously, it will be incubated for Composition is incubated at 37 DEG C +/- 1 DEG C.Preferably, will be incubated for composition be incubated at this kind of raised temperature at least 2 hours with Make antigenic stimulus immunocyte and generates immune effector molecule.Incubation step can be 2-50 hours, such as 2-40 hours, 5-30 Hour, 8-24 hours, the period between 16-24 hours or following values: 3,4,5,6,7,8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、 40,41,42,43,44,45,46,47,48,49 or 50 hours.In some embodiments, optional initial mixing step is being carried out Suddenly it antigen, non-reducing sugar and sample to be entirely incubated in composition after distribution, is carried out in the case where not mixing further It is incubated for.
In being incubated for composition, non-reducing sugar exists with certain concentration, and the concentration increases non-reducing sugar effectively respectively Strong antigen is stimulated to the stimulation of the cell of immune system and again.Therefore, non-reducing sugar is added with certain concentration, the concentration makes In positive sample (there is immunoreactive sample to antigen), compared with the case where being not added with non-reducing sugar, to incubation The level that non-reducing sugar improves the immune effector molecule (preferably interferon gamma) of generation is added in composition.Therefore, with spy Determine concentration addition non-reducing sugar, the concentration makes non-reducing sugar enhance immune effector molecule response magnitude, and display is thin at this time More immune effector molecule is produced in the sample for the immune response that born of the same parents mediate.Preferably, it is used with certain concentration non-reduced Sugar, the concentration make non-reducing sugar that immune effector molecule response is enhanced at least 1.1 times, preferably at least 1.2 times, more preferably At least 1.3 times.According to one embodiment, which maintains the immunocyte for including in sample within the extended period Mediate the ability of each response.According to one embodiment, the concentration for being incubated for non-reducing sugar in composition is at least 1mg/ml, at least 1.5mg/ml, preferably at least 1.75mg/ml, more preferably at least 2mg/ml.Exemplary range include but is not limited to 1mg/ml extremely 20mg/ml, 1.5mg/ml are to 17.5mg/ml, 2mg/ml to 15mg/ml, 3mg/ml to 15mg/ml, 4mg/ml to 12.5mg/ml With 5mg/ml to 10mg/ml.As shown in the Examples, these ranges are for example suitable for trehalose.It is non-reduced that it is also applied for other Sugared (such as sucrose, mannitol and gossypose), as shown in the Examples.According to one embodiment, it is incubated for non-reducing sugar in composition Concentration range be 1.5mg/ml to 10mg/ml, such as 1.75mg/ml to 7.5mg/ml or 2mg/ml to 5mg/ml.It can also be by Technical staff follows introduction described herein to determine suitable concentration.
One or more other additives can be added and therefore be included in and be incubated in composition.For example, can add One or more pairs of sample preparations and/or sample save required or advantageous additive, such as suitable anticoagulant is (if sample Product are blood samples).Preferably, which is heparin.Additive should not be can interfere cell-mediated immune response Concentration includes.According to one embodiment, other than non-reducing sugar, other monosaccharide are not added into incubation composition.According to one A embodiment does not add other reduced sugars into incubation composition other than non-reducing sugar, especially reduction monosaccharide.
According to one embodiment, the composition comprising antigen and non-reducing sugar is contacted the sample with.The embodiment is special It is not advantageous, because user need not individually add non-reducing sugar to being incubated in composition.This kind of ready-to-use composition is provided to keep away Exempt from operating error and saves the operating time.Optionally, diluent or solvent are included in containing antigen and non-reducing sugar In composition.In addition, may include one or more additives in the composition, on condition that it includes in being incubated for composition. The additive should not interfere with cell-mediated response.According to one embodiment, the composition also includes anticoagulant, preferably Heparin.According to one embodiment, the composition does not include monosaccharide.According to one embodiment, the composition does not include reduction Sugar, specifically it does not include reduction monosaccharide.
It is incubated for composition for preparation, is made comprising antigen, non-reducing sugar and optionally comprising one or more other additives The composition of (such as including anticoagulant in the case where blood sample) contacts sample.Sample can be added into composition, it is on the contrary ?.It is incubated for composition for preparation, preferably mixing sample, antigen, non-reducing sugar and other additive (if present)s.
The example of suitable composition forms includes liquid composition, semi-liquid composition, gel-form composition and solid Composition, especially dry composition.According to one embodiment, will add comprising antigen, non-reducing sugar and optional other The composition of agent is added to be included in collection containers, preferably sample collection tube, such as blood collection tube.This be particularly conveniently, Because sample directly contacts composition after collection.According to one embodiment, will comprising antigen, non-reducing sugar and it is optional its The composition spray of his additive is dry to the inside of collection containers.Spray drying process be in the prior art known to, Therefore any detailed description is not needed herein.
According to one embodiment, which is obtained from object and before contact non-reducing sugar and/or antigen not by such as group Knit culture, medium, excipient or the dilution of other liquid reagents.According to one embodiment, which includes at least 10 volume % samples.Term " at least 10 volume % " include sample volume be it is total be incubated for composition volume 10,11,12,13, 14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、 39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、 64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、 89,90,91,92,93,94,95,96,97,98,99 and 100 volume %.
As shown in the Examples, addition non-reducing sugar unexpectedly increases releasing for immune effector molecule in step a) It puts, such as especially interferon gamma, to improve the sensitivity of test.The effect is very surprising, because so far Until it is believed that the effect may only be reached by monosaccharide and therefore by reduction monosaccharide.It moreover has been found that comprising antigen and Even if the composition of non-reducing sugar also shows improved storage stability at elevated temperatures.Using observed by reduced sugar The effect that assay sensitivity and quality weaken at any time is not observed when using non-reducing sugar.Therefore, the present invention passes through offer Sensitive and stable storing test makes significant contribution to the prior art, and the test also maintains during extended storage Experimental performance.The teachings of the present invention advantageously allows to provide antigen and non-reducing sugar in the form of the composition of stable storing.? In the present invention, which is not used as the stabilizer of antigen.Show that antigen (especially peptide antigen) is to store up very much by experiment It deposits stable.Therefore, if storing antibody there is no sugar, it would not observe that the decline of experimental performance.Therefore, should Non-reducing sugar is for enhancing assay sensitivity, especially by enhancing immune effector molecule (especially cell factor, such as interferon Generation and/or release γ).
Step (b)
In step (b), detect whether that there are immune effector molecule or its horizontal raisings.As described above, there is (packet Include and be not present) immune effector molecule or its level instruction object in for test antigen cell-mediated immune response water Flat or ability.Specifically, the method allow to determine the object previously whether contacted test antigen or display with The antigen (pathogen such as to be detected) of test has the antigen of cross reactivity.It is thus possible to determine whether object can draw Hair respectively for the antigen, test antigen representated by antigen, pathogen or disease cell-mediated immune response.
The detection of immune effector molecule can occur in peptide or protein level or nucleic acid level, especially immune effector molecule MRNA expression.Therefore, immune effector molecule " whether there is or it is horizontal " is detected including directly or indirectly data.For example, Appropriate detection method (such as ELISA or ELISpot) directly measurement immune effector molecule can be used to whether there is or its content.So And in one embodiment, it whether there is based on rna expression horizontal measurement immune effector molecule or it be horizontal.It is high-caliber Immune effector molecule mRNA is the horizontal raised indirect data of display immune effector molecule.For measuring target gene mRNA expression Horizontal appropriate method be in the prior art known to, there is no need to any detailed descriptions.Therefore, in some embodiments, can make With ligand or binding molecule (as specificity is directed to the antibody of effector molecule) or the gene by measuring encoding immune effector molecule Expression detect immune effector molecule.
Immune effector molecule to be detected can be responsive cell activation, antigenic stimulus or stimulate generated a certain model again Enclose any one of molecule.Equally, more than one that can be discharged after test sample and the antigen contact of test in step (b) are exempted from The mode of epidemic disease effector molecule or immune effector molecule.Immune effector molecule to be measured can be produced by immunocyte, especially It can be produced by lymphocyte, the lymphocyte is such as T cell, especially CD4+T helper cell and/or CD8+Cell toxicant Property T cell.Therefore, in some embodiments, cell (especially T cell) of this method based on measurement immune system is to antigen Stimulation responses and the one or more immune effector molecules generated.However, nonimmune cell can also reply antigen to immune respectively It the stimulation of cell and stimulates again and discharges immune effector molecule, due to the thorn of its immune effector molecule discharged by immunocyte Swash, the stimulation of the immune effector molecule (such as IFN-γ) of the T cell release especially stimulated again.These immune effector molecules Information source can be important.Therefore, according to one embodiment, immune effector molecule to be detected can be thin by effect T Born of the same parents' response antigen stimulates again and the direct effect molecule that generates.In other embodiments, downstream immune effector molecule is measured. For example, can be measured in step (b) by immunocyte (especially T cell) IFN- produced for antigen (again) stimulation tested γ or other direct immunization effector molecules.However, as described above, these molecules usually induce or enhance other cells to generate it His immune effector molecule.The generation of these other (downstream) immune effector molecules can also be measured in step (b).The present invention also wraps Including the detection in step (b) is more than a type of immune effector molecule.According to one embodiment, in step (b) individually Or detect that immune effector molecule mode whether there is or it is horizontal together with direct immunization effector molecule (such as IFN-γ).Each mode Including more than two kinds, preferably greater than three kinds of different immune effector molecules.Analyzing each mode can provide having for object-immunity state Value information.For example, the mode of specific immune effector molecule or immune effector molecule can be the feature of specified disease.
According to one embodiment, immune effector molecule to be measured in step (b) is cell factor, as lymphokine, Interleukin or chemotactic factor (CF).Interferon (IFN) (such as IFN-γ) is particularly useful as immune effector molecule to be determined.Immunological effect Other examples of molecule include but is not limited to a certain range of cell factor, such as interleukin (IL), as IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, IL-13, IL-16 (LCF) or IL-17, IL-1 (IL-1F1), IL-1 β (IL-1F2), IL-1r α (IL-1F3), tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β), colony stimulating factor (CSF) are (such as Granulocyte (G)-CSF or granular leukocyte macrophage (GM)-CSF), complement component 5a (C5a), Gro α (CXCL1), sICAM-1 (CD54)、IP-10(CXCL10)、I-TAC(CXCL11)、MCP-1(CCL2)、MIF(GIF)、MIP-1α(CCL3)、MIP-1β (CCL4), silk suppression albumen E1 (PAI-1), RANTES (CCL5) or MIG (CXCL9).In some embodiments, the present invention provides Method, wherein in step (b) immune effector molecule to be detected be cell factor, it is the component of complement system, perforin, anti- Imperial element, tubulin (cathelicidin), granzyme, FasL, CD-40 ligand, exotoxin, cytotoxin, chemotactic factor (CF) or list Nuclear factor.In a preferred embodiment, the immune effector molecule detected in step (b) is IFN-γ.Therefore, according to preferred Embodiment, the present invention provides a kind of method of immune response cell-mediated in measurement object, the method includes will For sample collection from the object into collection vessel, the sample includes IFN-γ can be generated after by antigenic stimulus The sample and antigen and non-reducing sugar are incubated for by the cell of immune system, and then measurement whether there is IFN-γ or it is horizontal Raising, wherein there are IFN-γ or its level to indicate ability that described object establishes cell-mediated immune response.
The combination of immune effector molecule can be also detected in step (b).Therefore, step (b) specifically includes detection to antigen Stimulation responds and discharges and be the immune effector molecule (especially cell factor) of the feature of disease or illness to be analyzed Or the combination of immune effector molecule.In addition, the level of one or more immune effector molecules can other biological alone or in combination Marker or disease indicant are screened.
According to one embodiment, immunological effect point is detected by using the ligand of specific binding immune effector molecule Son.The ligand of immunoeffectors is particularly useful to detection and/or these quantitative molecules.It can be removed before detecting immune effector molecule It is incubated for the cell in composition included.It is known in the art for can be used for the technology for detecting test of step (b), and is wrapped It includes, such as radioimmunoassay test, sandwich assay, ELISA and ELISpot.The antibody of immunoeffectors is particularly useful as matching Body.Refer to that " antibody " includes the part (such as Fab segment) for specifically binding the antibody of immune effector molecule, mammalization (example Such as humanization) antibody, goes immune antiboidy, recombinantly or synthetically antibody and hybridization and single-chain antibody.Polyclonal and monoclonal antibody It can all be obtained with immune effector molecule or its antigen fragment are immune, and any kind can be used in immunity test.Obtain two types The method of type antibody is all well known in the art.Polyclonal antibody is less preferable, but is relatively easy to prepare, by suitable reality The immunoeffectors or its antigen part for testing room animal injection effective dose are collected serum from animal, and are exempted from any of Epidemic disease adsorber technologies separate specific serum.Although the antibody of such method production can be used for almost any type of immunoassay, But they usually because product potential heterogeneous and fall from favor.It is particularly useful using monoclonal antibody in immunoassay, Because its can mass production and product there is homogeney.It can be used technology well known to those skilled in the art thin by fusion immortality The born of the same parents system lymphocyte sensitive with to immunogenic preparation is prepared to obtain for the hybridoma cell line of monoclonal antibody production Object.Antibody for specific immune effector molecule is also commercially available.
According to one embodiment, step (b) includes making to be incubated for composition or part thereof (such as its portion for consuming cell Point) be enough to form antibody-effector compound for the antibody of immune effector molecule to be detected or its segment with specificity Under the conditions of contact a period of time, then detect the compound.As described above, incubation group can be removed by centrifugation before detection Close the cell for including in object.For example, when using blood as sample before detection, the life with immune effector molecule can be incubated for Cell is separated from incubation composition after producing and discharging, to provide plasma sample substantially.
Panimmunity experimental technique can be used, referring to U.S. Patent number 4,016,043,4,424,279 and 4,018,653. It can be combined with cell-mediated immune response test described herein to detect each test of the immune effector molecule of generation and also describe In WO2004/042396, WO2008/113119, WO2010/009494 and WO2011/075773, it is incorporated by reference this Text.In addition, " the Assays for monitoring cellular immune responses to active such as Clay Immunotherapy of cancer (test for monitoring the cellullar immunologic response of effective immunization therapy to cancer)； Clinical Cancer Research 2001；7:1127-1135 " describes several for monitoring cellullar immunologic response Method, to also illustrate the suitable examination for detecting the immune effector molecule generated in the response stimulated antigen (again) It tests.Wherein, test, ELISpot test and the test based on nucleic acid for example based on ELISA are described, real-time quantitative is such as passed through RT-PCR measures cytokine mRNA levels.Optionally, immunological effect is measured in the rna expression level based on immune effector molecule It, can be by the expression of the data normalization of acquisition to crt gene (such as CD8) when molecular level.Each method can be also combined with the present invention To detect the immune effector molecule generated.According to one embodiment, having used whether there is for detecting immune effector molecule Or its horizontal test based on nucleic acid.It can be used well known standard method in the prior art from incubation composition or its cell portion Nucleic acid (especially RNA) is separated in point.Preferably, the test based on amplification is used (to be preferably based on PCR's in this embodiment Test) come detect immune effector molecule whether there is or its expression raising.Can before amplification first using specificity for Isolated RNA reverse transcription is cDNA by the primer and/or probe of detection immune effector molecule.Preferably, which is quantitative. A kind of suitable method is quantitative real-time RT (reverse transcription) PCR.
Preferably, the detection of immune effector molecule is quantitative detection in step (b).
Particular implementation
Described hereafter is components used in the specific and preferred embodiment of the method for the invention and its.
As described above, in step (a), one or more other additives may include being incubated in composition.According to one A embodiment can add reagent to being incubated in composition with the activity of regulating regulatory T cell (T-reg cell).The latter's packet Include the inhibition function of inhibiting regulatory T cells.The reagent for the adjusting T-reg cell covered herein includes but is not limited to that CD25 matches Body, the justice for encoding JAK1 or TYK2 inhereditary material or antisense oligonucleotides, the CpG comprising oligonucleotides, are used as neutralizing antibody The oligonucleotides and other TLR regulators of toll sample receptor (TLR) regulator.In certain embodiments, T-reg cell is living Property be conditioned agent inhibition immune response inhibit cell." CpG molecule " refers to the oligonucleotides including CpG sequence or motif.T-reg The inhibitor of function or the example of regulator include CD25 ligand, the polyclonal or monoclonal antibody of including but not limited to CD25 or Its antigen-binding fragment, the humanization of CD25 or goes Immune polyclonal or monoclonal antibody or polyclonal or monoclonal antibody Recombinantly or synthetically form.Other examples of reagent include justice or antisense nucleic acid (nucleic) and molecule, for coding The micromolecular inhibitor of Janus tyrosine kinase 1 (JAK1) or tyrosine kinase 2 (TYK2) or JAK1 or TYK2 albumen MRNA or DNA.It refers to that " small molecule " includes immunoglobulin neoantigen receptor (IgNAR), is such as described in International Patent Publication No. WO 2005/118629.However, other examples of suitable agent include stimulant, for example, by Toll-like receptor (TLR) and/or The CpG molecule of other mechanisms.Therefore, this is also formed as the CpG comprising one or more oligonucleotides of TLR regulator A part of invention.Single type reagent can be used or carry out regulating regulatory T cell using two or more type agents.Example Such as, which can be carried out with following reagents: CD25 ligand and JAK1/TYK2 justice or antisense oligonucleotides；CD25 ligand and TLR Adjust reagent；JAK1/TYK2 justice or antisense oligonucleotides and TLR adjust reagent；Or CD25 ligand, JAK1/TYK2 justice or Antisense oligonucleotides and TLR adjust reagent.Alternatively, only using a type of reagent.Alternatively, can be used comprising oligonucleotides and The CpG of TLR regulator.Each T reg regulator can be separately added in sample and/or incubation composition or this may include containing In the composition for having antigen and/or non-reducing sugar.
According to one embodiment, the method for the invention includes
(a) by making the whole blood sample contact obtained from people's object include at least one peptide antigen and at least one non-reduced two The composition of sugared (preferably trehalose or sucrose) incubates the incubation composition in room temperature temperatures above to prepare incubation composition It educates at least 2 hours；And
(b) detect whether that there are IFN-γ or its levels；
Wherein, the level for cell-mediated immune response in object of leting others have a look at there are IFN-γ or its water-glass.
Preferably, which is antigen of the specificity for disease or pathogen.This document describes the non-limits of pathogen Property example processed.According to one embodiment, which is virus and this method allows to measure whether people's object has the ability to establish Antiviral cell-mediated immune response.
According to one embodiment, this method further includes
(c) compare detection immune effector molecule is horizontal or numerical value derived therefrom and reference levels.
Step (c) includes the immune effector molecule of comparative measurements horizontal or numerical value derived therefrom and reference levels.The reality The mode of applying is used in particular for pathogenic infection representated by diagnostic antigen.If the object is immunized by pathogenic infection, measurement Effector molecule is horizontal or numerical value derived therefrom is higher than reference levels, and if the object not by pathogenic infection, measurement Immune effector molecule level or numerical value derived therefrom are lower than reference levels.Identical principle is suitable for whether determining the object The cell-mediated immune response for pathogen representated by antigen can be established.
According to one embodiment, sample is divided at least two components and makes the first component of sample in step (a) Contact antigen and non-reducing sugar are negative right to generate to generate response sample and the second component of sample is made to contact inactive solution According to (nil) sample.In preferably the step of (b), determined whether in two kinds of components there are immune effector molecule or its It is horizontal.In step (c), surveyed by being deducted in negative control sample from the immune effector molecule level measured in response sample Fixed immune effector molecule level come measure sample antigen rely on immune effector molecule response.Then the antigen is relied on Immune effector molecule response or numerical value derived therefrom are compared with reference levels, to assist in whether object has previously contacted Antigen.It is thus possible to for example determine object whether fight the former risk for generating immune response, whether having generation disease and/or Whether may be by pathogenic infection.
Optionally, this method further includes that sample is divided at least three components and swashs the third component of sample with T cell Agent (such as mitogen) living is incubated for generate positive control sample.Herein, by immunocyte in such as three kinds independent groups Be incubated for: negative control sample (such as salt water), the response sample of antigenic stimulus and positive control sample (are combined using such as T cell Agent, such as mitogen, such as phytohaemagglutinin).Therefore, according to one embodiment, sample is divided at least three groups Point.In step (a), so that the first component of sample is contacted antigen and non-reducing sugar to generate response sample, make the second of sample Component contacts inactive solution to generate negative control sample and make the third component contact stimulus solution of sample (including, for example, promoting Mitogen) to generate positive control sample.In preferably the step of (b), determines whether to exist in three kinds of components and exempt from Epidemic disease effector molecule or its level.In step (c), by deducting yin from the immune effector molecule level measured in response sample Property control sample in the immune effector molecule level that measures measure the immune effector molecule response that the antigen of response sample relies on And the immune effector molecule response for relying on the antigen or numerical value derived therefrom are compared with reference levels；By from positive control The immune effector molecule level that measures is deducted in negative control sample in the immune effector molecule level measured in sample to measure Immune effector molecule response that the antigen of positive control sample relies on and by the response of gained immunological effect and reference levels or source Numerical value in it compares.It is thus possible to determine whether object previously contacted the antigen of test or the antigen of display and test The antigen of immune response is generated with cross reactivity and thereby to the antigen.Whether active for determining such as object, Whether whether recent or latent infection or object generate response to treatment, infection or disease whether will occur or exempt from What epidemic disease inhibited, this provides valuable help.For example, if the immune effector molecule response that antigen relies on, which is higher than, refers to water It is flat, then it can be the positive by outcome evaluation, i.e., object can generate the cell-mediated response for the antigen.If antigen according to Immunological effect response of the bad immune effector molecule response lower than reference levels and positive control dependence is higher than reference levels, then may be used It is feminine gender by outcome evaluation, i.e., object can not generate the cell-mediated response for the antigen.
It can the mitogen as positive control include in the present invention all rush divisions well known by persons skilled in the art Original and including but not limited to phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharides (LPS) and Phytolacca acinosa promote Mitogen (PWM).Other immunostimulant examples in addition to mitogen that can be used for providing positive control include but unlimited In chemical compound (such as R848).
As described above, in some embodiments, the container that wherein sample, antigen and non-reducing sugar are incubated for altogether is also to use In the collection vessel for collecting the sample from object.A large amount of different any one of used vessels can be used, on condition that its Suitable sample size is provided.In some embodiments, which is pipe, and it includes vacuum to promote the sample from object The collection of (such as blood).Each vacuum blood collecting tube is well known in the prior art, therefore is not needed herein any detailed Description.In other embodiments, which is capillary.In some embodiments, capillary is for through capillary action Blood is collected from skin surface.In some embodiments, sample is collected from object to containing at least one antigen, at least one In kind of non-reducing sugar and the collection vessel of at least one anticoagulant (preferably heparin), or antigen, non-is then added thereto Reduced sugar and anticoagulant (preferably heparin).In some embodiments, using capillary sampler (such as needling device) to blood Liquid be sampled and by the collection vessel of blood collection to test tube of hepari and be subsequently transferred to appropriate containers with antigen and it is non-also Raw sugar is incubated for altogether.Preferably, the form of single composition described herein above provides antigen, non-reducing sugar and optional anticoagulation Agent.In some embodiments, the whole blood collection from object is extremely contained into antigen, non-reducing sugar and optional anticoagulant In container.In other embodiments, antigen, non-reducing sugar and/or anticoagulant are added into whole blood sample after collection.
Exposure the invention is particularly useful for screening to pathogen, especially mycobacteria, such as mycobacterium tuberculosis.Therefore, Present invention teaches a kind of active method of immune response cell-mediated in measurement object, this method includes making comprising can be The sample contact mycobacteria specific antigen and nonreducing sugar of the immunocyte of immune effector molecule are generated after antigenic stimulus. Peptide as antigen includes all or part of mycobacterium tuberculosis protein antigen.After incubation, measures and exempt from caused by immunocyte The level of epidemic disease effector molecule (preferably interferon gamma) is wherein directed to tuberculosis branch bar in the horizontal instruction object of immune effector molecule The cell-mediated immune response of bacterium is horizontal.
ESAT-6 is the mycobacterium tuberculosis Early insulin secretion antigenicity target of 6kDa a kind of.ESAT-6 albumen (Early insulin secretion Antigenic target 6) it is the Major Secretory antigen purified from mycobacterium tuberculosis Short-term Culture filtrate.As described herein, ESAT-6, CFP-10 (culture filtrate protein 10) and 85B are purified available from cell lysate and by recombinant technique or to close At peptide form production.For example, ESAT6 can be obtained from national serum research institute (SSI, Denmark's brother's sheet in the form of recombinant protein Breathe out root).Other suitable target protein antigens of mycobacterium tuberculosis include TB7.7 and TB37.6.CFP10 is also referred to as ESAT-6 The antigenic protein MTSA-10 of sample albumen eesxB and secretion.
Tuberculin or PPD (protein derivatives of purifying) be different from ESAT-6 albumen (Early insulin secretion antigenicity target 6), CFP-10 (culture filtrate protein 10) and TB7.7, by the base being only located in mycobacterium tuberculosis gene group (in the area RD-I) Because encoding and being not included in BCG (BCG vaccine).It is different from PPD, because PPD also contains and such as BCG sub-strain and several tools Other antigens for having the non-tuberculous mycobacteria kind of low pathogenicity or no pathogenicity shared.According to one embodiment, PDD by with Make antigen.Specifically, the protein derivatives (PPD or tuberculin) of terms used herein purifying are a kind of non-species specificities The sediment of molecule.By extracting albumen from mycobacterium tuberculosis or the compound of other mycobacterias (such as mycobacterium avium) To obtain PPD or tuberculin.PPD is commonly used in testing whether to exist generating for BCG or for mycobacterium tuberculosis thin Born of the same parents are immunized or Thl response.For example, it is available from Connaught Laboratories Ltd. (Connaught Laboratories Limited TubersolB) is prepared arrogant masterbatch (Master Batch), Connacht's tuberculin (CT68), either RT23 form obtained from national serum research institute (SSI, Copenhagen, Denmark).
Preferably, which is selected from CFP10, ESAT-6, TB7.7 and TB37.6 from mycobacterium tuberculosis.According to one A embodiment provides antigen by corresponding to these proteantigens and/or showing the peptide to its cross reactivity.
In one embodiment, CD4 is contacted the sample with+And CD8+The combination of peptide, is described in detail above.We With reference to foregoing disclosure.
After the extended period after object blood drawing, the cell loss of immune system increases cell-mediated in whole blood immune answer The ability answered, and glitch-free response is usually seriously reduced or is lacked after blood drawing for about 24 hours.It works and in the present invention The reduction of the demand of special equipment makes it possible to carry out cell-mediated immune response stimulation, the medical treatment using antigen in medical center Point is on such as doctor clinic, clinic, outpatient service facility and veterinary inspector or farm.Once antigenic stimulus is completed, it is no longer necessary to new Fresh and competent cell.IFN-γ and other cell factors for being discharged due to antigenic stimulus or immune effector molecule cell-free or Remove cell fluid such as blood plasma in stablize, therefore, sample can with for other infectious diseases or other diseases diagnosis Standard plasma or the similar mode of blood serum sample are stored or are transported without specific condition or rapid time requirement.Therefore, excellent The immune effector molecule actually discharged is surveyed in Selected Inspection.However, include be incubated for composition or the full cell being incubated in composition can be It is contacted after incubation with the nucleic acid stability composition comprising stablizing rna expression mode, on condition that the RNA table based on immune effector molecule It whether there is up to level measurement immune effector molecule or it be horizontal.Several stable compositions are commercially available.For example, PAX is public (PreAnalytiX) is taken charge of to provide containing the composition for the reagent of rna gene expression overview in fast and stable blood.Each combination Object can also be used to stablize the rna gene expression overview for being incubated for the cell in composition included.Each stable composition allows in room temperature Lower transhipment and storage induce and transcribe without Yin Jiyin risk that degradation causes RNA overview to change (see, for example, US 6,617, 170, US 7,270,953, Kruhoffer etc., 2007).Each composition is with PAXgene blood rna pipe (PAXgene Blood RNA Tubes) title sell.
Using, disease and illness
Non-limiting application, including purposes, disease and illness is described below.Obviously, the method for the invention and sheet The text composition and kit can be widely used in medical treatment and diagnostic field and for example can be used for thin suitable for analysis patient The in vitro test for the response that born of the same parents mediate.In addition, the method for the invention and composition described herein and kit are valuable Analysis tool, for test agent (such as Immunotherapeutic agent for cancer) stimulation and therefore enhance cell-mediated immune ability.
The method instructed herein can whether there is disease or illness or its horizontal or stage in such as test object, described Disease or illness are such as causative agent infection, autoimmune disease, cancer, are exposed to inflammatory agent, are exposed to drug, are exposed to Toxic protein reagent and for example by disease symptom induce or by Chemicals induction immune deficiency or immuno-suppressive conditions.It is reliable and Delicately the ability of measurement cell-mediated immunity is important for such as evaluation object ability, and the ability is to causative agent The response of (such as microorganism, virus or helminth) infection establishes autoimmune response, to vaccine or immunotherapeutic agent response, guarantor Shield resists cancer or other neoplastic conditions, detection inflammatory conditions or test object to the sudden and violent of toxic agent (such as beryllium or environmental agent) Dew or sensibility.Test described herein keeps the early detection of immune response power and/or more sensitive detection feasible.Examination described herein Testing also to promote and facilitate to detect causes immunosuppressive disease symptom or detection by drug-induced immunosupress.Therefore, originally " the cell-mediated immune response in measurement object " that text is instructed has many useful applications in medical field, hereinafter will Non-limiting purposes and application are described.
For example, methods described herein can be used for infectious or autoimmune disease immunodiagnosis, as immunocompetent Marker and inflammatory disease, allergen, cancer, immunotherapeutic agent effect marker, and the label as toxic agent Object.In addition, this method is commonly used in detection for t cell response (including the measurement vaccine effect of endogenous and/or exogenous antigen Rate).
Based on cell, functional immunity response test is also accepted as influencing vaccine, the immunization therapy of immune response Efficient surrogate markers object in agent and the exploitation of biological agent.The test can be used for academic environment, pharmaceutical environment and vaccine and The research and development of biological agent.The assessment of immune cell function ability is for understanding some diseases illness and for its treatment It is most important for tactful efficiency.Immunocyte is responsible for preventing or be treated, such as in the infectious diseases of similar HIV, or Responsible disease symptom itself, such as in autoimmune disease illness.It can be used the method for the invention in autoimmune disease Autoantigen-specificity reaction is measured in (such as multiple sclerosis) patient.The present invention provides tests for these purposes.
Kinds cancer Immunotherapy Strategy is tested in clinical test at present.Although clinical efficiency be these methods most Test eventually, but the very long and complicated development approach of these projects is needed to assess and be marked as among most possible successfully candidate Remember the immune response of object.This address need accurately to detect and quantify T cell mediation, the test of antigen-specific immune response. For each immunotherapeutic agent (it is for example not necessarily expected to lead to tumor regression, but still has beneficial effect to disease), it is necessary to base In it is assumed that active patterns select biomarker.For immunization therapy, this kind of marker is can be by one or more immune examinations Test the stimulation of the specific for tumour antigen immune response of detection.Herein, it is preferable to use test assessment CD8+Cytotoxic T is thin Born of the same parents and the CD4 for causing cytotoxic T cell to generate+The function of T helper cell (especially T assists 1 type response), the CD8+Carefully The tumour peptide for the direct cell cracking of triggering that MHC molecule is presented on cytotoxic T cells Direct Recognition tumor cell surface.This hair It is bright to provide the test that can be used for the purpose because of its sensitivity and reliability.Therefore, methods described herein, kit and group Close object can be used for analyzing a kind of medicament (such as tumor therapeutic agent) whether can enhance it is cell-mediated immune.This, which can be used, to obtain (such as standardized) immunocyte (example is as described above) tested in such as aggregate level, or can also be in single patient Middle test is situated between to analyze specific immunotherapeutic agent (such as Immunotherapeutic agent for cancer) and whether can enhance cell in the patient That leads is immune.This kind of test Curing circumstance for clinical development and later is valuable, thus analyze patient whether by Beneficial to using the treatment of immunotherapeutic agent.The example of immunotherapeutic agent includes but is not limited to biological agent, and it is (special such as to enhance T cell It is not cytotoxic T cell) active therapeutic antibodies.Binding mode is incoherent, on condition that it causes or may cause Cell-mediated immune stimulation/increase.For example, can be by directly stimulating immunocyte or by being reduced or switched off negative regulation institute The active suppression mechanism of T cell is stated to enhance activity, to stimulate/increase cell-mediated be immunized indirectly.Another example is Her skin wood monoclonal antibody (Ipilimumab) of cancer immunotherapy antibody.
In addition, the method for the invention can be used in test object whether there is disease or illness or its horizontal or stage, Wherein, immune effector molecule is detected the presence of using the method for the invention or its level indicates the disease or illness.
In addition, the method for the invention can be used for measuring reagent or disease symptom whether induce immunosupress in object or It is associated therewith, wherein to detect the presence of immune effector molecule or its level instruction using the method for the invention and induced by reagent Or by disease symptom induction or immunosuppressive degree relevant to disease symptom.
One aspect of the present invention includes measuring the response for specific antigen by using the method for the invention to show Show the method for immune response cell-mediated in object.In one embodiment, sample (the leucocyte group of such as whole blood, enrichment Point or BAL fluid) available from suffer from or may occur specified disease (such as autoimmune disease, causative agent infect Or be exposed to disease caused by toxic agent) object, and exempted from by using the first aspect of the present invention the method to measure Epidemic disease response, such as response is generated from effector T cell (such as CD4 to antigenic stimulus by detection+T cell and/or CD8+Cell toxicant Property T cell) in discharge immune effector molecule.
Method of the invention be used in particular for disease or illness in detection and/or monitoring object (infection of such as causative agent, itself Immunological diseases, cancer or inflammatory disease), level or stage including the disease or illness.Other illnesss includes being exposed to toxicity Reagent (such as beryllium).Test of the invention can also be used to monitor therapeutic scheme.
Cell is situated between in the presence of the immune effector molecule generated in the antigen response to test or its level instruction object The level for the response led.Specifically, the horizontal instruction of response is presence or absence of disease or illness or its horizontal or stage, institute It states disease or illness is selected from causative agent infection, autoimmune disease, cancer, inflammatory conditions and is exposed to toxic agent.It is specific and Speech, there are disease or illness or its water representated by immune effector molecule or its antigen of level instruction presence or absence of test The flat or stage.
This method can also be used in the response of the therapeutic scheme in monitoring object to disease or illness.There are the immune effects of generation The effect of answering molecule or its horizontal or mode that can indicate therapeutic scheme.
The method of the present invention also can referred to as " be tested ".This method is a kind of ex vivo approach.Test described herein is for evaluation pair The common immune response of elephant or for detecting the immune response to specified disease illness, the disease symptom is such as autoimmunity Disease, chylous diarrhea, cancer, pathogenic organisms or reagent infection, be exposed to toxic agent or drug and immune deficiency or immune suppression Illness processed (such as by disease symptom or therapeutic-induced).
In one embodiment, object is people and cell-mediated immune response test is for screening to micro- life of causing a disease Object, virus and the response of helminth, development potentiality, or monitoring autoimmune disorder, chylous diarrhea, monitored object are to tumor challenge Response and determine whether that there are any immune deficiency or immunosupress.The latter can be by some drugs for example including various chemotherapeutics Cause.Alternatively, the exposure to environment toxic agent and pollutant can be tested.In one embodiment, immunosuppressive illness is caused Including chronic infection and cancer.Can lead to immunosuppressive other illnesss includes inflammatory disorders.It can lead to immunosuppressive drug Including for treat those of rheumatic arthritis, cancer and inflammatory bowel disease drug or with organ transplant combination those of medicine Object.
In one embodiment, methods described herein can be used for auxiliary diagnosis or monitoring may with tuberculosis (such as Active, latent or recent TB infection) patient and be especially developed to the active raised patient of tuberculosis risk from latent, such as Receiving immunosuppressive drug, (i.e. (anti-CD 20 antibodies are (such as mab treatment) or TNF-α blocking treatment (such as))) or steroids or cancer chemotherapy patient；Alternatively, with immune suppression Illness processed (such as HIV infection, cancer, IDDM or Non-Insulin Dependent Diabetes Mellitus (NIDDM), autoimmune disease, nutrition are not Good, aging, intravenous drug use (IVDU) or heredity immunologic derangement) patient, and recently infected individual.
In one embodiment, methods described herein can be used for monitoring has infection or other diseases illnesss after diagnosing Object.This can for example facilitate the therapeutic efficiency during or after assessment treatment terminates, such as by monitoring and predicting possibility Infection and recurrence.
According to one embodiment, whether this method is infected for measure object and/or can establish for anti- The cell-mediated immune response of pathogen representated by original.
Pathogen or infectious agent include bacterium, helminth and virus.The example of bacterium includes Gram-positive and gram Negative germs, such as Mycobacterium (Mycobacterium), staphylococcus (Staphylococcus), streptococcus (Streptococcus), Escherichia coli (Escherichia coli), salmonella (Salmonella), clostridium Belong to (Clostridium), Shigella (Shigella), Proteus (Proteus), bacillus (Bacillus), Hemophilus (Hemophilus), primary Shu Shi Spirochaeta (Borrelia) etc..Mycobacterium tuberculosis (Mycobacterium Tuberculosis) and the illness such as tuberculosis (TB) of mycobacterium tuberculosis infection generation is particularly useful target.Disease Malicious example includes that hepatitis virus (hepatitis type B virus and Hepatitis C Virus), herpesviral and CMV virus and human immunity lack Fall into virus (HIV) and its caused disease.Helminth includes Plasmodium (Plasmodium), ring tinea, liver helminth etc..Its His pathogen includes eukaryocyte, such as yeast and fungi.Evaluation is exposed to potential or actual thin in the object for infecting entity The response that born of the same parents mediate is usually extremely important.Method of the invention can also be used for detecting whether that there are these infection and disease mistakes The level of journey or stage.
This method, which can also be used in monitoring, to be had in the people that each disease risks occur for the thin of certain diseases and/or infectious agent The immune level that born of the same parents mediate.Another example is the cmv infections in immunosuppressive patient (such as Organ Transplantation Patients).CMV sense Dye occurs usually in the form of immunosupress complication, especially after the transfer, and significantly leads to the illness in transplant recipient And death.Currently used for preventing the immunosuppressive therapy of transplant organ rejection to T lymphocyte and cell-mediated immune response With illeffects, lead to a possibility that transplanting restrovirus infection rising.CD8+CMV specificity cell toxicity T lymphocyte (CTL) the fact that virus associated-diseases occur can be protected against also to highlight T cell function and inhibit the importance of CMV duplication. Another example of immunosuppressed patient is the patient of HIV infection.Method of the invention can be used for having each disease wind of generation The level that disease immune (such as anti-CMV is immune) is successively monitored in the people of danger, because the forfeiture of the immune function can be with disease (such as CMV disease) development correlation.Preferably, interferon gamma is measured in each test as effector molecule.The immune state of transplant recipient It can influence CMV (again) activation in transplant recipient.For example, the strong jamming element γ response that CMV specific antigen is induced indicates CMV The risk of disease reduces, because patient has strong cell-mediated immune response and is therefore protected from virus infection.It is minimum Interferon gamma response indicate raised CMV disease risks because being not present or there are low-down cell-mediated immune.Number According to display, after antiviral prevention stops, compared with the patient with negative test, the patient tested with positive CMV significantly compared with Continually and more long maintain do not have CMV disease.Therefore, compared with not having those of detectable immune response patient, Preventing latter stage has the significantly lower risk that CMV disease occurs with the patient of cellullar immunologic response to CMV.Therefore, this hair The development that Delayed onset CMV disease in transplant recipient can be predicted in bright the method is simultaneously therefore highly useful to case control.
Alternatively, the object can be tested with following disease symptom or for following disease symptom: chylous diarrhea, self Immunity diabetes, alopecia areata, ankylosing spondylitis, anti-phospholipid syndrome, autoimmunity Addison's disease multiple sclerosis Disease, adrenal gland autoimmune disease, autoimmune hemolytic anemia, auto immune hepatitis, autoimmunity oaritis and orchitis, White Sai Shi (Behcet's) disease, epidermolysis class day bleb, cardiomyopathy, sprue dermatitis (celiac sprue- Dermatitis), chronic fatigue syndrome (CFIDS), chronic inflammation demyelinate, chronic inflammation polyneuropathy, that Qiu-applies Er Shi is comprehensive Close disease, cicatricial pemphigoid, CREST syndrome, cold coagulation disease, Crohn's disease, dermatitis herpetiformis, plate-like erythema wolf Sore, elementary mixing cryoglobulinemia, fibromyalgia, glomerulonephritis, Gray's Freund disease, guillain-Barre disease, tall Ben's Thyroiditis, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephrosis, insulin-dependent glycosuria Sick (I type), lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, the heart Myositis, pemphigus vulgaris, pernicious anaemia, nodular polyarteritis, polychondritis, polyadenous body syndrome, polymyalgia rheumatica, Polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Lei Nuoshi are existing As, fiessinger leroy syndrome, rheumatic fever, rheumatoid arthritis, meat-like tumor disease, chorionitis, Sjogren syndrome, stiff people's syndrome, Systemic loupus erythematosus, high iS-One arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, blood vessel Scorching, hickie and inflammatory bowel disease.
The object can suffer from cancer or can be tested for cancer.Treatment of cancer depend in part on cell-mediated immunity and Tumour itself or drug for treating tumour can lead to immunosupress.Tumour described herein includes one group of disease and disorder, Feature is unregulated cell growth (such as tumour is formed) and these cells are not divided into any special and different cell.These Disease and disorder include: ABL1 proto-oncogene, AIDS associated cancer, acoustic neurinoma, acute lymphatic leukemia, acute marrow Chronic myeloid leukemia, adenoid cystadenocarcinoma, adrenocortical carcinoma, agnogenic myeloid metaplasia, alopecia, maceration sarcomatous tissue, cancer of anus, Angiosarcoma, alpastic anemia, neuroastrocytoma, ataxia telangiectasia, basal-cell carcinoma (skin Skin), bladder cancer, osteocarcinoma, intestinal cancer, brain stem glioma, brain and cns tumor, breast cancer, cns tumor, carcinoid tumor, cervix cancer, youngster Virgin brain tumor, childhood cancer, leukemia of children, children soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic Leukaemia, colorectal cancer, skin T cell lymphoma, dermatofibrosarcoma protuberans, promotees fiber group at chronic myelocytic leukemia Knit Hypertrophic small cell carcinoma, duct carcinoma, endocrine cancer, carcinoma of endometrium, ependymoma, cancer of the esophagus, ewing's sarcoma, liver outer bladder Road cancer, cancer eye (eye cancer), eye: melanoma, retinoblastoma, carcinoma of fallopian tube, Fanconi anemia, fiber meat Tumor, gallbladder bladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal associated cancers tumour, the cancer of urogenital system, gonioma, gestation are grown Support cell disease tumour, glioma, gynecological cancer, Hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hereditary breast cancer, histocytosis, Hodgkin's disease, human papilloma virus, water pocket shape mole, hypercalcinemia, under Pharynx cancer, intraocular melanoma, islet-cell carcinoma, Kaposi sarcoma, kidney, cell of Langerhan histocytosis, larynx cancer, Leiomyosarcoma, leukaemia, Li-Fo Meini syndrome, lip cancer, embryonal-cell lipoma, liver cancer, lung cancer, lymphedema, lymthoma, The pernicious Rhabdomyosarcoma of Hodgkin lymphoma, non-Hodgkin lymphoma, male breast carcinoma, kidney, medulloblastoma, melanoma, Merkel's cells cancer, celiothelioma, metastatic carcinoma, carcinoma of mouth, MEN,muitiple endocrine neoplasms, mycosis fungoides, myeloproliferative disorder are comprehensive Close disease, myeloma, myeloproliferative disease, CARCINOMA OF THE NASAL CAVITY, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, neurofibroma Disease, Nijmegen breakage syndrome, nonmelanoma skin cancer, non-small cell lung cancer (NSCLC), cancer eye (ocular Cancers), cancer of the esophagus, carcinoma of mouth, oropharyngeal cancer, osteosarcoma, neostomy oophoroma, cancer of pancreas, nasal sinus cancer, accessory thyroid glands cancer, the cheek Adenoncus tumor, carcinoma of penis, peripheral neuroectodermal tumour, pituitary tumor, polycythemia vera, prostate cancer, rare cancer With related disease, clear-cell carcinoma, at retinoblastoma, rhabdomyosarcoma, congenital poikiloderma, Salivary Gland Tumors, Sarcoma, neurinoma, Sai Zhali syndrome, cutaneum carcinoma, Small Cell Lung Cancer (SCLC), intestinal tumor, soft tissue sarcoma, spinal cord are swollen Tumor, gastric cancer, synovial sarcoma, carcinoma of testis, thymic carcinoma, thyroid cancer, transitional cell carcinoma (bladder), is moved squamous cell carcinoma (skin) Row cell cancer (kidney-renal plevis -/- ureter), choriocarcinoma, carcinoma of urethra, the cancer of urinary system, diaphragm plate block albumen, uterus meat Tumor, uterine cancer, carcinoma of vagina, carcinoma of vulva, Walden Si Telunshi macroglobulinemia and the nephroblastoma.
The exposure of albumen toxic agent is tested alternatively, the object can be exposed to albumen toxic agent or can be directed to.
Autoimmune disease described herein for detecting and/or monitoring includes alopecia alopecia areata, ankylosing spondylitis, anti-phosphorus Rouge syndrome, autoimmunity Addison's disease, multiple sclerosis, adrenal gland autoimmune disease, autoimmune haemolytic are poor Blood, lupoid hepatitis, autoimmunity oaritis and orchitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, stomatitis Property diarrhea dermatitis (celiac sprue-dermatitis), chronic fatigue syndrome (CFIDS), chronic inflammation demyelinate are chronic Inflammation polyneuropathy, allergic granulomatous angiitis, cicatricial pemphigoid, CREST syndrome, cold coagulation disease, clone Family name's disease, dermatitis herpetiformis, lupus erythematosus discoides, elementary mixing cryoglobulinemia, fibromyalgia, glomerulonephritis, lattice Thunder Freund disease, guillain-Barre disease, Hashimoto thyroiditis, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephrosis, insulin-dependent diabetes mellitus (I type), lichen planus, lupus erythematosus, Meniere disease, Combination connective group Knit disease, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anaemia, how soft nodular polyarteritis is Osteitis, polyadenous body syndrome (polyglancular syndromes), polymyalgia rheumatica, polymyositis and dermatomyositis, it is former Hair property agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, fiessinger leroy syndrome, rheumatism Heat, rheumatoid arthritis, sarcoidosis, chorionitis, Sjogren syndrome, holotonia syndrome, systemic loupus erythematosus are more Hair property aorto-arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis and leucoderma.
The other diseases illness of consideration includes inflammatory disease condition, because it can lead to immunosupress.It is by the invention The example of inflammation disease illness includes but is not limited to: cause some regions to redden, swelling, pain and fever feel response disease And disorder, it is used to protect and is damaged or the tissue of sickness influence.Can use the method for the present invention treat inflammatory disease include but It is not limited to: acne, angina pectoris, arthritis, aspiration pneumonia, disease, empyema, enterogastritis, inflammation, intestinal flu, NEC, necrosis Property enteritis, pelvic inflammatory disease, pharyngitis, PID, pleurisy, throat inflammation, redness, rubescent, sore throat, stomach flu and urinary tract infections, slow Property inflammation Demyelinating Polyneuropathy disease, chronic inflammation Demyelinating Polyneuropathy root neuropathy, chronic inflammation Demyelinating Polyneuropathy disease, chronic Inflammation Demyelinating Polyneuropathy root neuropathy.About non-human application, the present invention also detects the EIPH and various animal disorders of horse, example Such as Tasmanian devil facial tumors.
In above-mentioned aspect, the antigen is available from causative agent, the related or toxic agent to disease symptom or cancer.Alternatively, sense Dye, disease symptom, cancer or toxic agent mediation capable of inhibiting cell it is immune, can be used object previously to contact at this time any anti- It is former.
Hereinafter describing again especially has according to described in the specification and claims of the method for first aspect The embodiment of benefit.According in a first aspect, the present invention provides a kind of for measuring the active side of cell-mediated immune response Method, the method includes
(a) it is incubated by making the sample comprising immunocyte contact at least one antigen and at least one non-reducing sugar to provide Composition is educated, the immunocyte can generate immune effector molecule after being stimulated by antigen, and
(b) it detects whether to have at least one immune effector molecule or it is horizontal.
According to one embodiment, which is non-reducible disaccharide, is preferably selected from trehalose and sucrose.According to one Embodiment, the concentration for being incubated for non-reducing sugar in composition is at least 1.5mg/ml, preferably at least 2mg/ml.Above also describe It is incubated in composition the appropriate concentration range of non-reducing sugar and referring to above disclosure.
According to one embodiment, which is selected from trehalose, mannitol, sucrose and gossypose.Such as embodiment institute Show, these non-reducing sugars improve response level.Trehalose is particularly preferred, because observing that response is horizontal most herein It is big to increase.In addition, reinforcing effect can be retained during storage.
According to one embodiment, in step (a), the composition comprising antigen and non-reducing sugar is contacted the sample with.Root According to an embodiment, wherein sample is whole blood sample, and the composition also may include anticoagulant, preferably heparin.According to one Embodiment, the composition comprising antigen, non-reducing sugar and optional anticoagulant are included in collection containers.As above Described, this kind of collection containers may, for example, be evacuated blood collection container.According to one embodiment, the composition is spray The dry composition of mist.This document describes suitable embodiments.
According to one embodiment, which has one or more following characteristics:
I) sample is obtained from people's object；
Ii) sample is obtained from people's object of immunosupress or immune deficiency；
Iii) sample includes immunocyte, and the immunocyte is selected from NK cell, T cell, B cell, dendritic cells, huge Phagocyte and monocyte；And/or
Iv) sample is whole blood.
According to one embodiment, which is selected from peptide, albumen (including glycoprotein), phosphoprotein and phospholipoprotein, carbon water The segment of compound, phosphatide and aforementioned substances, and preferably provided by one or more peptides.
According to one embodiment, two or more not synantigen and/or inspections in step (b) are used in step (a) Survey two or more different effect molecules.
According to one embodiment, one or more peptides are used as antigen, length is selected from 5-100 amino acid or 7-50 A amino acid.According to an advantageous embodiment, the antigen is by one or more by CD8+The peptide of cytotoxic T cell identification It provides.Preferably, in this embodiment, which is selected from by one or more length less than 15 amino acid, preferred lengths The peptide of 7-14 amino acid provides.Described above is the details of each peptide and referring to above disclosure.
According to one embodiment, which is provided by least two groups peptide, and first group is about 7- comprising at least one length The peptide of 14 amino acid residues and second group are 15 amino acid residues or longer peptide comprising at least one length comprising complete Portion or a part of proteantigen.Described above is the details of each peptide group and referring to above disclosure.
According to advantageous embodiment, which is provided by one or more synthetic peptides.Described above is the details of peptide and Referring to each disclosure.
According to one embodiment, antigen that is related to disease or illness or representing disease or illness is contacted the sample with, The immune response that middle testing needle mediates the disease or illness test cell.According to an advantageous embodiment, the antigen It is disease-specific antigens, specifically pathogen specific antigen.For example, the antigen can come from and disease symptom The antigen of related pathogen or with it with cross reactivity, or tumor associated antigen relevant to cancer.Such as institute above It states, which can be bacterium, virus, helminth, yeast or fungi.Non-limiting embodiment is described below.For example, Bacterium can be selected from Gram-positive and gram-negative micro-organism, especially Mycobacterium (Mycobacterium) (such as tuberculosis Mycobacteria), staphylococcus (Staphylococcus), streptococcus (Streptococcus), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Clostridium (Clostridium), shigella dysenteriae Belong to (Shigella), Proteus (Proteus), bacillus (Bacillus), hemophilus (Hemophilus) With primary Shu Shi Spirochaeta (Borrelia).Virus can be selected from hepatitis virus (such as hepatitis type B virus and Hepatitis C Virus), Herpesviral, CMV virus and human immunodeficiency virus (HIV).Helminth can be selected from Plasmodium (Plasmodium), ring tinea With liver helminth.
According to one embodiment, which comes from or specificity is for virus, preferably cytomegalovirus (CMV).It is preferred that Ground, the antigen are provided by one or more peptides, and the length is 7-14 amino acid residue, 7-13 amino acid residue or 8-12 A amino acid residue.As described above, which can be synthetic peptide.
According to one embodiment, the immune effector molecule detected in step (b) has one or more following characteristics:
I) it is cell factor；
Ii) it is chemotactic factor (CF)；
Iii) its to the stimulation of cell-stimulating, antigen or again stimulation responses and generate；
Iv) its be selected from interferon, interleukin (IL), tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β), Colony stimulating factor (CSF), such as granulocyte (G)-CSF or granular leukocyte macrophage (GM)-CSF, complement component 5a (C5a), Gro α(CXCL1)、sICAM-1(CD54)、IP-10(CXCL10)、I-TAC(CXCL11)、MCP-1(CCL2)、MIF(GIF)、MIP-1 α (CCL3), MIP-1 β (CCL4), silk suppression albumen E1 (PAI-1), RANTES (CCL5) or MIG (CXCL9)；And/or
V) immune effector molecule is IFN-γ.
According to this method embodiment, immune effector molecule whether there is or its level is based on immune effector molecule It measures or the rna expression level based on immune effector molecule measures.Suitable embodiment is described above.
According to one embodiment, method described in any one of aforementioned embodiments is deposited for monitoring or determining whether In disease or illness or its horizontal or stage, the disease or illness are selected from causative agent infection, autoimmune disease, cancer, inflammation Venereal disease disease is exposed to toxic agent, to therapeutic agent response, immune deficiency and immunosupress.Preferably, cell-mediated immune to answer The magnitude answered is related to the state of disease symptom, progress and/or seriousness.According to one embodiment, in aforementioned embodiments Described in any item methods are for detecting or monitoring disease, infection and/or response to treatment, especially with immunosupress The immunization therapy or treatment that agent carries out.
According to one embodiment, method described in any one of aforementioned embodiments includes
(a) by making the whole blood sample contact obtained from people's object include at least one peptide antigen and at least one non-reduced two The incubation composition is incubated at least 2 hours by the composition of sugared (preferably trehalose or sucrose) to provide incubation composition；With And
(b) measurement is with the presence or absence of the IFN-γ discharged by antigenic stimulus or its level；
Wherein, there is the level for cell-mediated immune response in object of leting others have a look at through the IFN-γ detected or its scale.
According to one embodiment, method described in any one of aforementioned embodiments includes
(c) by the immune effector molecule level of measurement or numerical value derived therefrom compared with reference levels.
The present invention also provides a kind of method for the treatment of object, the object suffers from pathogenic infection, autoimmune disease Or cancer or the tendency with this kind of conditions or diseases of generation, this method include carrying out the party according to the first aspect of the invention Method is to measure cell-mediated response activity, wherein there is cell in immune effector molecule or its level instruction object after measured The response of mediation is horizontal, indicates whether conditions or diseases or its horizontal or state, then treats the conditions or diseases.On Text describes the details of first aspect the method and referring to above disclosure.
Composition, kit and purposes
According to the second aspect of the invention, the combination of the immune response mediated for inducing cell in the sample is provided Object, it includes
A) at least one isolated antigen；
B) at least one non-reducing sugar；
C) optional at least one anticoagulant.
It describes above and in the method for the invention and is related to composition, composition forms, suitable antigen, non-reduced Sugar and anticoagulant details and referring to above disclosure.This kind of composition can be used for method described in such as first aspect. Be described in detail above suitable application (including purposes/purpose, can be used the analysis of this kind of composition disease and illness and Therefore the Suitable applications of the composition) and referring to above disclosure.Composition described in second aspect is especially suitable for upper Literary the method, application and use.According to one embodiment, the composition does not include monosaccharide.According to one embodiment, should Composition does not include reduced sugar.Preferably, the composition is semiliquid, gel or solid composite.It preferably, is drying Composition.According to one embodiment, the composition is the composition of spray drying.
It is non-limiting, advantageous embodiment is described below again:
The non-reducing sugar can have above and feature described in the method for first aspect and referring to equally answering herein Each disclosure.As described above, which is not used as the stabilizer of antigen but for improving response level.Strictly according to the facts It applies shown in example, it is horizontal that non-reducing sugar (such as trehalose, mannitol, sucrose and gossypose) improves response.According to an embodiment party Formula, the non-reducing sugar are non-reducible disaccharides.The non-reducible disaccharide can be selected from trehalose and sucrose.According to one embodiment, When the concentration of non-reducing sugar contacts the sample of the composition and desired amount in the two aspect compositions, gained incubation group Closing object includes the non-reducing sugar that concentration is at least 1.5mg/ml, preferably at least 2mg/ml.Side described in above and first aspect Also described in method be incubated for composition in non-reducing sugar appropriate concentration range and referring to above disclosure.Above and first Suitable and preferred specimen material is also described in method described in aspect and referring to above disclosure.According to an embodiment party Formula, the sample have one or more following characteristics:
I) sample is obtained from people's object；
Ii) sample is obtained from people's object of immunosupress or immune deficiency；
Iii) sample includes immunocyte, and the immunocyte is selected from NK cell, T cell, B cell, dendritic cells, huge Phagocyte and monocyte；And/or
Iv) sample is whole blood.
According to one embodiment, the antigen for including in composition is selected from peptide, albumen (including glycoprotein), phosphoprotein and phosphorus The segment of lipoprotein, carbohydrate, phosphatide and aforementioned substances, and preferably provided by one or more peptides.
According to one embodiment, the composition includes two or more different antigens.
According to one embodiment, the composition includes that one or more peptides are used as antigen, one or more peptides Length is selected from 5-100 amino acid or 7-50 amino acid.According to an advantageous embodiment, the composition includes as anti- Former is one or more by CD8+The peptide of cytotoxic T cell identification.Preferably, in this embodiment, the antigen is by one kind Or less than 15 amino acid of different lengths, preferred length are selected from the peptide offer of 7-14 amino acid.Above and first aspect institute Suitable each peptide is described in the method stated and referring to above disclosure.
According to one embodiment, the composition includes the antigen provided by least two groups peptide, and first group includes at least one It comprising at least one length is 15 amino acid residues or longer that kind of length, which is the peptide of about 7-14 amino acid residue and second group, Peptide comprising all or part of proteantigen.Suitable each peptide is described above and in method described in first aspect Group and referring to above disclosure.
According to advantageous embodiment, the composition includes the peptide provided by one or more synthetic peptides.Described above is The details and each disclosure of reference of peptide.
According to one embodiment, the composition includes antigen related or representing disease or illness to disease or illness, The wherein testing needle immune response cell-mediated to the disease or illness.
It include antigen in composition is disease-specific antigens according to advantageous embodiment, it is specifically sick Pathogen-specific antigen.For example, the antigen can have friendship from the antigen from pathogen related to disease symptom or with it Fork reactivity, or tumor associated antigen relevant to cancer.As described above, which can be bacterium, virus, posts Infested, yeast or fungi.Non-limiting embodiment is described below.For example, the bacterium can be selected from Gram-positive and leather is blue Family name's negative germs, especially Mycobacterium (Mycobacterium) (such as mycobacterium tuberculosis), staphylococcus (Staphylococcus), streptococcus (Streptococcus), Escherichia coli (Escherichia coli), salmonella Belong to (Salmonella), Clostridium (Clostridium), Shigella (Shigella), Proteus (Proteus), bacillus (Bacillus), hemophilus (Hemophilus) and primary Shu Shi Spirochaeta (Borrelia).Virus can be selected from hepatitis virus (such as hepatitis type B virus and Hepatitis C Virus), herpesviral, CMV virus With human immunodeficiency virus (HIV).Helminth can be selected from Plasmodium (Plasmodium), ring tinea and liver helminth.
According to one embodiment, the composition includes to come from or specificity is for viral (such as cytomegalovirus (CMV)) Antigen.Preferably, the antigen is provided by one or more peptides, and the length is 7-14 amino acid residues, 7-13 amino Sour residue or 8-12 amino acid residue.As described above, which can be synthetic peptide.
The composition may include in collection containers, and there is described herein this kind of container, (such as evacuated blood collection holds Device) suitable and advantageous embodiment and referring to each disclosure.
The present invention also provides a kind of incubation compositions, include at least one isolated peptide, at least one non-reducing sugar, energy Enough immunocytes (preferably T lymphocyte) that immune effector molecule is produced after antigenic stimulus and optional at least one anticoagulation Agent.Preferably, which prepares from whole blood sample and therefore comprising whole blood sample.According to one embodiment, lead to Crossing makes the contact sample of composition described in second aspect prepare incubation composition.It retouches above and in method described in first aspect Suitable and preferred specimen material is stated and referring to above disclosure.According to one embodiment, the sample have it is a kind of or A variety of following characteristics:
I) sample is obtained from people's object；
Ii) sample is obtained from people's object of immunosupress or immune deficiency；
Iii) sample includes immunocyte, and the immunocyte is selected from NK cell, T cell, B cell, dendritic cells, huge Phagocyte and monocyte；And/or
Iv) sample is whole blood.
Preferably, by making the contact of composition described in second aspect of the present invention be obtained from the whole blood of object (being preferably obtained from people) Sample obtains incubation composition.Above and composition described in the method for the invention and second aspect is described about second The aspect composition is incubated for composition, its preparation, the details of suitable antigen, non-reducing sugar and anticoagulant and referring to upper State disclosure.The anticoagulant is preferably heparin.According to one embodiment, it is at least which, which includes concentration, The non-reducing sugar of 1.5mg/ml, preferably at least 2mg/ml.Also illustrate incubation group above and in method described in first aspect Close object in non-reducing sugar appropriate concentration range and referring to above disclosure.According to one embodiment, which selects From trehalose, mannitol, sucrose and gossypose.As described above, according to advantageous embodiment, which is non-reduced Disaccharides is preferably selected from trehalose and sucrose.
A kind of collection containers, especially sample collection tube are additionally provided, it includes each composition, the composition packet Contain
A) at least one isolated antigen；
B) at least one non-reducing sugar；
C) optional at least one anticoagulant.
It include that composition in collection containers is preferably composition described in second aspect.Described above is about Composition, suitable antigen, the details of non-reducing sugar and anticoagulant and referring to above disclosure.This kind of collection containers It can be used for method described in such as first aspect.Be described in detail above suitable application (including purposes/purpose, can be used The disease and illness of this kind of collection containers analysis) and referring to above disclosure.Sample collection described in the third aspect is held Device is especially suitable for method as described above, application and use.Preferably, composition is sprayed into container inside, the container is excellent Choosing is vacuum blood collecting tube.By using this kind of instant collection vessel in the method for the invention, it can optimize and standardize The condition of this method simultaneously simplifies operation.
It is preferable to use kits to carry out the method for the invention, material needed for which provides execution method and step Material.This kind of kit preferably comprises standardized material, ensures that this method carries out under optimal conditions, so that it is guaranteed that being obtained from Different samples or patient or the result obtained by different operating personnel are comparable between each other.Therefore, in fourth aspect In, the present invention also provides the active kits of immune response cell-mediated in a kind of measurement object, and it includes at least one Antigen, at least one non-reducing sugar, at least one collection containers and at least one are for detecting at least one immunological effect The detection means of molecule.Preferably, the antigen and non-reducing sugar are provided in the form of single composition.To this end it is possible to use, this Composition described in invention second aspect and for the details of the composition referring to above disclosure.It is above and of the invention The details for being related to composition, antigen and non-reducing sugar is described in the method and composition and referring to above disclosure.Root According to an embodiment, which includes collection containers (such as blood collection tube), and it includes contain antigen and non-reduced The composition of sugar.Preferably, the composition also includes anticoagulant (such as heparin).As described herein, sample collection is included in hold Composition in device can be composition described in second aspect.Preferably, which is immunologic function test reagent, is such as marked Antibody.However, the detection means can be by specificity for be detected immune for the test based on detection mRNA expression The primer and/or probe of effector molecule provide.Suitable test and detection means are known to the skilled in the art and equally As described above.
According on the other hand, the present invention relates to for measuring non-in the active immunity test of cell-mediated response go back The application of raw sugar, wherein the addition of sample and antigen incubation period non-reducing sugar increases immune effector molecule in immunocyte Release, the immunocyte generates response to the antigen tested in the test.
Above and the method for the invention describes and is related to non-reducing sugar, preferred concentration, immune in medical field Test and its application, the details of suitable and preferred antigen, immune effector molecule and immunocyte and referring to above disclosure. The non-reducing sugar can have one or more following characteristics:
I) non-reducing sugar is disaccharides.
Ii) non-reducing sugar is selected from trehalose and sucrose；
Iii) non-reducing sugar is with the concentration of at least 1mg/ml, preferably 2mg/ml to contain sample to be tested and antigen It is incubated in composition；And/or
Iv) non-reducing sugar provides in the form of compositions, and the composition also includes antigen to be tested and optionally resists Coagulant.
According to one embodiment, which is selected from trehalose, mannitol, sucrose and gossypose.
Non-reducing sugar (such as trehalose) described above is for measuring or monitoring answering in cell-mediated immune test With with significant advantage.It is preferable to which ground, provides non-reducing sugar in the form of the single composition contacted with sample And antigen.According to one embodiment, addition non-reducing sugar contains non-reducing sugar for incubation and/or non-reducing sugar In the composition of antigen.As described above, which is not used as the stabilizer of antigen.According to advantageous embodiment, The antigen is by one or more by CD8+The peptide (preferably synthetic peptide) of cytotoxic T cell identification provides.Preferably, in the implementation In mode, which is selected from the peptide of 7-14 amino acid (preferably by less than 15 amino acid of one or more length, preferred length Synthetic peptide) it provides.
According on the other hand, the present invention relates to described in composition, third aspect present invention described in second aspect of the present invention Kit described in collection containers and/or fourth aspect present invention is for measuring in the active test of cell-mediated response Application.The details for being related to suitable and preferred purposes and application is described above and referring to above disclosure.According to one Embodiment, the test are for monitoring or determining whether that there are disease or illness or its horizontal or stage, the disease or diseases Disease be selected from causative agent infection, autoimmune disease, cancer, inflammatory conditions, be exposed to toxic agent, to therapeutic agent response, be immunized Defect and immunosupress.According to one embodiment, which is for detecting or monitoring disease, infection and/or to treatment Response, the immunization therapy or treatment carried out especially with immunosuppressor.According to one embodiment, second aspect of the present invention Kit described in collection containers described in the composition, third aspect present invention and/or fourth aspect present invention is used for In first aspect the method described in text detailed description and claim.
The present invention is not limited to illustrative methods disclosed herein and materials.Numberical range described herein includes limiting the range Numerical value.Title provided herein is not the limitation of various aspects or embodiment of the invention, can be whole with reference book Understand various aspects or embodiment of the invention.According to one embodiment, described herein includes certain steps (for method For) or theme comprising certain ingredients (for such as composition, solution and/or mixture) refer to by each step or ingredient The theme of composition.Preferably select and combine preferred embodiment described herein and combined by each preferred embodiment formed it is specific Theme also belongs to the present invention.
The present invention is described in further detail below by the mode of non-limiting embodiment.
Embodiment
Embodiment 1: shadow of the trehalose for Epstein epstein-Barr virus (Epstein-Barr virus) assay sensitivity It rings
It carries out measuring answering to the model antigen EBNA-1 from Epstein epstein-Barr virus using QuantiFERON technology The increase for the assay sensitivity that non-reducing sugar is induced is added in the research answered to assess into mixtures incubated.In short, to blood EBNA-1 peptide, 3 concentration (0.5,1.0 or 2.0mg/ml trehalose) of plus/minus aqueous trehalose are added in liquid collecting pipe.According to Manufacturer's specification measures the QuantiFERON test of anti-EBV cellullar immunologic response.It is measured and is compared according to trehalose concentration Compared with IFN-γ response.The increase of IFN-γ response is observed using increased trehalose concentration.The sea 2mg/ml in the testing experiment The concentration of algae sugar provides optimum.
These studies have shown thats, which add non-reducing sugar (such as trehalose) into whole blood test, leads to cell-mediated immune response IFN-γ response dramatically increases in test.By the increase for the IFN-γ response that addition non-reducing sugar is induced, examination is enhanced The sensitivity tested.
Embodiment 2: the storage stability of prior art test
In this embodiment, the storage for testing and analyzing test component for the cell-mediated immune response of CMV is carried out Stability.As antigen, having used length is the synthetic peptide from cytomegalovirus related antigen of 8-12 amino acid.Containing Have and prepares CMV correlation synthetic peptide in the solution of monosaccharide (glucose) and be sprayed on the inside of blood collection tube and dried. Therefore, antigen and monosaccharide are included in a composition.It include the stability test of the composition in blood collection tube Test activity declines at any time when display stores at room temperature.In addition, that will include the spraying drying containing antigen and glucose After the blood collection device of composition stores more than the time of 3 weeks (21 days) at 55 DEG C, the test loss of activity of CMV device. Therefore, each composition and the collecting pipe comprising each composition are not suitable for instant kit group due to lacking storage stability Point.Stability and function that glucose has restored test are removed from composition.It therefore, in the composition include monosaccharide and antigen It is infeasible.On the contrary, to increase assay sensitivity, it is necessary to which into sample, individually addition is single during preparation is incubated for composition Sugar.
On the contrary, the composition comprising antigen and non-reducing sugar teaching herein can also be tieed up during extended storage Hold increased assay sensitivity.
These results are also by other experimental verifications.By the CMV pipe containing glucose or without glucose in 4 DEG C, 22 DEG C, 37 DEG C and 55 DEG C at store three weeks and the blood sample from 4 reactive donors and 1 non-reacted donor then used to carry out Test.The average result that the display of table 1 is obtained using reactive donor.At shown temperature, the reaction of the CMV pipe containing glucose Property shown lower than the pipe stored at 4 DEG C or 22 DEG C and relative to the liquid preparation (reference standard) for being used to prepare CMV pipe it is significant Response decline.On the contrary, the pipe for lacking glucose does not show that each reactivity is lost.
Table 1: the influence of glucose during storage.Show the fold difference (average IU/ml) to reservation
55℃ 37℃ 22℃ 4℃
CMV pipe containing glucose 0.58 0.75 1.48 1.34
CMV pipe without glucose 0.97 0.91 1.01 1.21
Influence of the trehalose for IFN-γ response is added in embodiment 3:QFN-CMV pipe
QuantiFERON (QFN) CMV is a kind of test of CE registration, and monitoring is for from the anti-of cytomegalovirus Former T cell immunological memory.QFN-CMV blood tube contains peptide, and it is dry to generate which is designed to specific activation CD8+T cell Disturb plain γ (IFN-γ).These pipes only contain peptide and heparin, do not add any glycan molecule.
The experiment is intended to study influence when adding non-reducing sugar trehalose into QFN-CMV pipe for IFN-γ response.
Trehalose (water) solution of 0,1,5 or 10mg/ml is added into QFN-CMV blood collection tube.Then to this four Middle 1ml blood of the addition from 17 healthy donors with known anti-CMV t cell response of each of pipe, furthermore uses Nil pipe and mitogen pipe are as control.QFN test is then carried out according to package insert.All numerical value all deduct Nil value.Needle To IFN-γ level of each QFN-CMV+0mg/ml trehalose (untreated control) standardization as unit of IU/ml to generate ' again Number variation ' unit；Difference between donor to control response magnitude.Including not having the health that can detect anti-CMV immune response Donor is controlled with the non-specific IFN-γ for trehalose induction.
Addition trehalose cause the concentration dependent of IFN-γ average level increase (be expressed as IFN-γ compare it is matched not The multiple variation of processing control).Dramatically increasing for effect is observed when adding 5 and 10mg/ml trehalose；It is examined using Friedman It tests and Dunn multiple comparative test.Shown in the result is shown in Figure 1.IFN-γ background level in CMV negative control donor is not observed Dramatically increase (data are not shown).
Into QFN test, addition trehalose is significantly improved to the specific antigen response contained in blood collection tube and is produced Raw IFN-γ is horizontal, horizontal without non-specifically improving background IFN-γ.
Influence of the trehalose for IFN-γ response is added in embodiment 4:QFN-TB pipe
To confirm, into the test (such as QuantiFERON (QFN) test) of assessment peptide specific cellular immunity, addition is non-also The observation of the quantitative result of raw sugar trehalose enhancing test, manufacture are managed added with the QFN of non-reducing sugar.Manufacture QFN blood collection Pipe is to contain the synthetic peptide from mycobacterium tuberculosis (MTB) antigen ESAT-6 and CFP-10, wherein not adding any sugar or adding Add non-reducing sugar trehalose.
It is come from using the test of established QuantiFERON platform technology and is tried using QuantiFERON Gold In Tube Testing confirmation has a whole blood of 20 objects of MTB infection sign, but using it is described above through manufacture do not have sugar (without sugar) or The QFN TB antigen pipe of modification with non-reducing sugar (trehalose).According to be have been established the QFT Gold package insert of test into Row test.In the pairs of clinical analysis, determining for test is significantly enhanced comprising non-reducing sugar (trehalose) in TB antigen pipe It measures response (P=0.0005), as shown in Figure 2.Numerical value (y-axis) from pipe is with the shape of the IFN-γ IU/ml numerical value of Logarithm conversion Formula shows and (has deducted nil pipe numerical value).List tail t in pairs is carried out to examine to show the statistical significance between two groups.As a result clear Ground shows that addition trehalose increases interferon gamma response, as shown in the object infected with TB.
Embodiment 5: different non-reducing sugars are added and increase quantitative IFN-γ response
The reinforcing effect observed is also shown using other non-reducing sugars.For the experiment, to QFN CMV antigen pipe and phase Four kinds of different non-reducing sugars are added in the Nil pipe answered.For the final haemoconcentration for reaching 2mg/ml, to being obtained from 5 donors The PBS solution of corresponding sucrose, trehalose, mannitol and gossypose is added in whole blood.Therefore, to the blood from 5 donors QFN test is carried out, wherein using non-reducing sugar (N/S) or addition non-reducing sugar (sucrose, trehalose, mannitol or cotton is not added Sub- sugar) QFN CMV pipe.According to being that the QFN package insert of test has been established to be tested.As a result as shown in Figure 3.X-axis (Fig. 3) Show the condition of test.It background correction (numerical value of the Nil pipe from each sugar of add/not add) and is being counted from corresponding CMV pipe The percentage of IFN-γ numerical value (IU/ml) increases in the non-adding tube (y-axis) calculated.The results show that using all four non-reduced Sugar all observed the increased CMV antigen response measured by quantitative IFN-γ (IU/ml).Therefore, the non-of all tests is gone back Raw sugar all has beneficial effect to IFN-γ response.Highest increase is observed when using trehalose, is mannitol, sucrose later And gossypose.

Claims (22)

1. non-reducing sugar is preparing the application in the product for measuring the active method of cell-mediated immune response, the side Method includes:
(a) incubation group is provided by making the sample comprising immunocyte contact at least one antigen and at least one non-reducing sugar Object is closed, the immunocyte can generate immune effector molecule after being stimulated by antigen；And
(b) it detects whether to have at least one immune effector molecule or it is horizontal, wherein the non-reducing sugar is trehalose；
The product further includes antigen, and the length of the antigen is selected from 5-100 amino acid.
2. application as described in claim 1, the concentration for being incubated for non-reducing sugar described in composition is at least 1.5mg/ml.
3. application as described in claim 1, the concentration of the non-reducing sugar is at least 2mg/ml.
4. the application as described in one or more in claim 1-2, in step (a), the sample contact is comprising described anti- The composition of the former and described non-reducing sugar.
5. application as claimed in claim 4, the composition are included in collection containers.
6. application as claimed in claim 4, the composition also includes anticoagulant, and the sample is whole blood sample.
7. application as claimed in claim 6, the anticoagulant is heparin.
8. application as described in claim 1, the sample have one or more following characteristics:
I) sample is obtained from people's object；
Ii) sample is obtained from people's object of immunosupress or immune deficiency；
Iii) sample includes immunocyte, and the immunocyte is selected from NK cell, T cell, B cell, dendritic cells, macrophage Cell and monocyte；And/or
Iv) sample is whole blood.
9. application as described in claim 1, the antigen have one or more following characteristics:
I) one or more peptides are used as antigen, and the length of one or more peptides is selected from 5-100 amino acid or 7-50 Amino acid；
Ii) antigen is provided by one or more synthetic peptides；
Iii) antigen is provided by least two groups peptide, and first group is about 7-14 amino acid residue comprising at least one length Peptide and second group are 15 amino acid residues or longer peptide comprising at least one length, and the peptide includes all or part of egg Bai Kangyuan；
Iv) antigen is by by CD8+One or more peptides of cytotoxic T cell identification provide；And/or
V) antigen is by by CD8+One or more peptides of cytotoxic T cell identification provide and one or more peptides Length is less than 15 amino acid.
10. application as claimed in claim 9, in v), the peptide length is selected from 7-14 amino acid.
11. application as described in claim 1, wherein the antigen that has used two or more different in step (a) and/or Two or more different effector molecules are had detected in step (b).
12. application as described in claim 1, the antigen is disease-specific antigens.
13. application as claimed in claim 12, the disease-specific antigens are pathogen specific antigen.
14. application as claimed in claim 1 or 8, the antigen has one or more following characteristics:
I) antigen is related to disease or illness or represents disease or illness, wherein being directed to the disease or illness test cell The immune response of mediation；
Ii) antigenic source in the antigen from disease symptom correlation pathogen or with it with cross reactivity, either Tumor associated antigen relevant to cancer；
Iii) antigen is pathogen specific antigen and the pathogen is bacterium, virus, helminth, yeast or fungi；
Iv) antigen is pathogen specific antigen, i.e. bacterium specific antigen, and the bacterium be selected from Gram-positive and Gram-negative micro-organism；
V) antigen is pathogen specific antigen, i.e. viral-specific antigens, and the virus is selected from hepatitis virus, bleb Virus, CMV virus and human immunodeficiency virus (HIV)；And/or
Vi) antigen comes from or specificity is for virus, and is provided by one or more peptides, and the length of the peptide is 7-14 Amino acid residue, 7-13 amino acid residue or 8-12 amino acid residue.
15. application as claimed in claim 14, iv) in bacterium be selected from Mycobacterium, staphylococcus, streptococcus, Escherichia coli, Salmonella, Clostridium, Shigella, Proteus, bacillus, hemophilus With primary Shu Shi Spirochaeta.
16. application as claimed in claim 15, the bacterium of the Mycobacterium is mycobacterium tuberculosis.
17. application as claimed in claim 14, v) in hepatitis virus be selected from hepatitis type B virus and Hepatitis C Virus.
18. application as claimed in claim 14, vi) in virus be cytomegalovirus (CMV).
19. application as described in claim 1 is used to monitor or determine whether that there are disease or illness or its horizontal or ranks Section, the disease or illness are selected from causative agent infection, autoimmune disease, cancer, inflammatory conditions, are exposed to toxic agent, are right Therapeutic agent response, immune deficiency and immunosupress.
20. application as described in claim 1, which comprises
(a) by making to provide obtained from composition of the whole blood sample contact comprising at least one peptide antigen and trehalose of people's object It is incubated for composition, and the incubation composition is incubated at least 2 hours；And
(b) measurement is with the presence or absence of the IFN-γ discharged by the antigenic stimulus or its level；
Wherein, there is the level that immune response cell-mediated in people's object is indicated through the IFN-γ detected or its amount.
21. non-reducing sugar answering in the product for preparing the method for measuring the active immunity test of cell-mediated response The non-reducing sugar is added with, wherein sample and antigen incubation period increasing, the antigen tested in the test is produced The immune effector molecule of the immunocyte release of raw response, the non-reducing sugar is trehalose.
22. application as claimed in claim 21, the non-reducing sugar have one or more following characteristics:
I) non-reducing sugar is at least concentration of 1mg/ml to contain the incubation group of sample to be tested and the antigen It closes in object；And/or
Ii) non-reducing sugar provides in the form of compositions, and the composition also includes antigen to be tested and optional anticoagulant Blood agent,
Wherein the length of the antigen is selected from 5-100 amino acid.
CN201380065249.4A 2012-12-28 2013-12-20 Cell-mediated immune response test Active CN105051537B (en)

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